Compositions and Methods for Treating Non-Alcoholic Steatohepatitis

TECHNICAL FIELD

The present invention relates to compositions and methods comprising ethyl icosapentate for treatment of non-alcoholic steatohepatitis (NASH).

BACKGROUND ART

It is known that heavy alcohol use can lead to liver complications, including alcoholic hepatitis which is often characterized by fatty liver and inflammation. Alcoholic hepatitis can ultimately lead to cirrhosis of the liver (scarring) and hardening of the liver tissue.

Individuals that do not consume excessive amounts of alcohol can also be found to have liver disease complications. Non-alcoholic fatty liver disease (NAFLD) is understood to encompass a variety of liver diseases, including steatosis (simple fatty liver), non-alcoholic steatohepatitis (NASH) and advanced scarring of the liver (cirrhosis). NASH has traditionally been diagnosed by means of a liver biopsy to characterize the liver histology, particularly with respect to the characteristics of inflammation, fibrosis and steatosis (fat accumulation). NASH then generally prefers to clinical findings based upon the liver biopsy of a patient with steatohepatitis, combined with the absence of significant alcohol consumption (Neuschwander-Tetri, B. A. and S. H. Caldwell (2003) Hepatology 37(5): 1202-1209). In NASH, fat accumulation is seen in varying degrees of inflammation (hepatitis) and scarring (fibrosis). Patients having NASH are also often characterized by abnormal levels of liver enzymes, such as aspartate aminotransferase (AST, GOT) and alanine aminotransferase (ALT, GPT). However, a clinical diagnosis of NASH still depends upon a liver biopsy to assess the histologic characteristics of the patient's liver, such that histological examination of liver biopsy tissue is often characterized as the “gold-standard” technique for the assessment of liver fibrosis (Neuschwander-Tetri, ibid).

CITATION LIST
Non Patent Literature

- Non Patent Literature 1; Hepatology June 2005; 41:1313-1321 “Design and validation of a historical scoring system for nonalcoholic fatty liver disease”

SUMMARY OF INVENTION
Technical Problem

The object of the present invention is to provide the compositions and methods comprising ethyl icosapentate for the treatment or alleviation of non-alcoholic steatohepatitis (NASH), and alleviation of the symptoms associated with NASH.

Solution to Problem

In one embodiment of the invention is that a pharmaceutical agent for treatment or alleviation of symptoms of non-alcoholic steatohepatitis (hereinafter abbreviated as NASH), an effective amount of ethyl icosapentate is administered after determining in a subject a baseline level indicative of NASH of at least one criteria selected from the group consisting of NAS score, steatosis score, lobular inflammation score, ballooning score and fibrosis stage.

In (1) embodiment of the invention Ethyl icosapentate for use in the treatment or alleviation of symptoms of NASH in a subject in need thereof, wherein:

(a) a baseline level in a subject having NASH of at least one criteria selected from the group consisting of NAS score, steatosis score, lobular inflammation score, ballooning score and fibrosis stage is determined; and

(b) an effective amount of ethyl icosapentate (EPA-E) is administered to said subject.

(2) The ethyl icosapentate for use of (1), wherein said subject has a NAS score of 4 or more than 4.

(3) The ethyl icosapentate for use (1) or (2), wherein said subject is characterized by at least one criteria selected from the group consisting of a baseline ALT value of 10 to 300 U/L; a baseline AST value of 10 to 250 U/L; a baseline steatosis grade of 2 to 3; and a baseline lobular inflammation grade of 2 to 3.

(4) The ethyl icosapentate for use of any one of (1) to (3), wherein after said administration of said EPA-E for about one year, said subject exhibits at least one improvement selected from the group consisting of a reduced ALT value as compared to said baseline ALT value; a reduced AST value as compared to said baseline AST value; a reduced steatosis grade as compared to said baseline steatosis grade; and a reduced lobular inflammation grade as compared to said baseline lobular inflammation grade.

(5) The ethyl icosapentate for use of any one of (1) to (4), wherein said ethyl icosapentate is administered to said subject in an amount of between 300 to 4000 mg per day.

(6) The ethyl icosapentate for use of any one of (1) to (5), wherein said subject is further characterized by having at least one condition selected from the group consisting of high TG, low HDL-C, diabetes, impaired glucose tolerance and metabolic syndrome.

(7) The ethyl icosapentate for use of any one of (4) to (6), wherein said reduced ALT value is at least 5% lower than said baseline ALT value and/or said reduced AST value is at least 5% lower than said baseline AST value.

(8) The ethyl icosapentate for use of any one of (1) to (7), further comprising determining in said subject prior to treatment a baseline level in serum of at least one member selected from the group consisting of ALT in a range of 10 to 300 U/L, AST in a range of 10 to 250 U/L, HDL-C in a range of 25 to 55 mg/dl, LDL-C in a range of 100 to 200 mg/dl, triglycerides in a range of 100 to 1000 mg/dl, TC in a range of 170 to 300 mg/dl, High TG and low HDL-C, TG/HDL-C ratio in a range of 3.75 to 10, non-HDL-C in a range of 100 to 250 mg/dl, Free fatty acid in a range of 400 to 1000 micro Eq/L, HOMA-IR in a range of 1.5 to 5, HbAlc in a range of 5.7 to 10%, Fasting plasma glucose in a range of 100 to 200 mg/dl.

(9) The ethyl icosapentate for use of any one of (1) to (8), wherein after administration of ethyl icosapentate for at least 3 months, said subject exhibits the following changes in said at least one marker as compared to the baseline level of at least 1% reduction for ALT, AST, TG, TG/HDL ratio, Free fatty acid, AA, MUFA, Palmitoleic acid, Oleic acid, Oleic acid/Stearic acid ratio, Palmitoleic acid/Palmitic acid ratio, Adrenic acid/AA ratio, Ferritin, Thioredoxin, TNF-alpha, sTNF-R1, sTNF-R2, Hs-CRP, CRGF, sCD40, Leptin, complement factor D, CK18 fragment, serum HMGB1, soluble Fas antigen, Hyaluronic acid, Type IV collagen (7s domain), procollagen III peptide or PAI-1; at least 5% increase for EPA or EPA/AA ratio; at least 1% increase for DPA, AA/Homo-gamma-linolenic acid ratio or Serum adiponectin; no worsening of ALP, bilirubin, GGT, Albumin, HDL-C, LDL-C, TC, non-HDL-C, HOMA-IR, HbAlc, Glucose, Fasting plasma glucose, postprandial plasma glucose, OGTT, platelet count or BMI.

(10) The ethyl icosapentate for use of any one of (1) to (9), wherein: the NAS score in said subject after administering (i) to a composite score of 3 or less than 3 and no worsening of said fibrosis stage score, or (ii) by 2 or more than 2 across at least two of the NAS components and no worsening of said fibrosis stage score is improved.

In another embodiment of the invention the ethyl icosapentate for use in the treatment or alleviation of symptoms of NASH, wherein an effective amount of ethyl icosapentate is administered to a subject for treating NASH after identifying the subject having NASH; determining the baseline level in the subject of at least one criteria selected from the group consisting of NAS score, steatosis score, lobular inflammation score, ballooning score and fibrosis stage.

In another embodiment of the invention ethyl icosapentate for use in the treatment or alleviation of symptoms of NASH, wherein a subject/patient having NASH is identified after determining the baseline level in the subject of at least one criteria selected from the group consisting of NAS score, steatosis score, lobular inflammation score, ballooning score and fibrosis stage; administering to the subject an effective amount of ethyl icosapentate; and improving the NAS score (i) to a composite score of less than 3 or equal to 3 or (ii) by 2 across at least two of the NAS components, combined with no worsening of the fibrosis stage score.

In another embodiment of the invention the ethyl icosapentate for use in treatment or alleviation of symptoms NASH, wherein the identification is a subject having NASH characterized by baseline levels of ALT of between 5 to 300 U/L and at least one criteria selected from the group consisting of NAS score of 4 or more than 4, a steatosis score of 1 or more than 1, a lobular inflammation score of 1 or more than 1 and either (i) a fibrosis stage of at least 1a or (ii) ballooning; administering to the subject an effective amount of ethyl icosapentate; and improving the NAS score in the subject (i) to a composite score of 3 or less than 3 or (ii) by 2 or more than 2 across at least two of the NAS components, together with no worsening of the fibrosis stage score.

In another embodiment of the invention, the ethyl icosapentate for use in treatment or alleviation of symptoms NASH, wherein;

a subject is identified having NASH characterized by baseline levels of ALT of between 5 to 300 U/L and at least one criteria selected from the group consisting of NAS score of 4 or more than 4, a steatosis score of 1 or more than 1, a lobular inflammation score of 1 or more than 1 and either (i) a fibrosis stage of at least 1a or (ii) ballooning, and at least one or any combination of two or more of the pretreatment baseline of the items mentioned in Tables 1 and 2;

a baseline level in blood or physical condition prior to treatment in the subject is determined;

an effective amount of ethyl icosapentate is administered to the subject; and the NAS score in the subject (i) to a composite score of 3 or less than 3, or (ii)

by 2 or more than 2 across at least two of the NAS components, together with no worsening of the fibrosis stage score, optionally improving at least one selected from the items mentioned in Tables 1 and 2 is improved.

In another embodiment of the invention, the ethyl icosapentate for use in treatment or alleviation of symptoms NASH, wherein the subject is taking at least one drug selected from the group consisting of lipid-lowering drugs, HMG-CoA reductase inhibitors (stains), fibrates, probucol, ezetimibe, ursodiol (UDCA), taurine, betaine, N-acetylcysteine, s-adenosylmethionine (SAM-e), milk thistle, anti-TUMOR NECROSIS FACTOR (TNF) therapies, probiotics, anti-diabetic medications, biguanides (metformin), insulin, sulfonylureas, alpha-glucosidase inhibitors (acarbose), dipeptidyl-peptidase 4 inhibitors (sitagliptin, saxagliptin, alogliptin, vildagliptin, linagliptin, etc.), phenylalanine derivatives (nateglinide, repaglinide), anti-platelet therapy, anti-thrombotic agents, Glucagon-like peptide-1 (GLP-1) receptor agonists (liraglutide, exenatide, taspoglutide, etc.), PDE-4 inhibitor, angiotensin II-1 type receptor antagonist (ARB: losartan, etc.), polyenephosphatidylcholine, antioxidant (vitamine E, vitamin C, nicotinic acid tocopherol, etc.), and pentoxifylline.

In another embodiment of the invention, ethyl icosapentate for use in the treatment or alleviation of symptoms of NASH wherein,

a subject is identifyed having NASH characterized by baseline levels of ALT of between 5 to 300 U/L and at least one criteria selected from the group consisting of NAS score of 4 or more than 4, a steatosis score of 1 or more than 1, a lobular inflammation score of 1 or more than 1 and either (i) a fibrosis stage of at least 1a or (ii) ballooning, and at least one or any combination of two or more of the pretreatment baseline of the items mentioned in Tables 1 and 2;

a baseline level in blood or physical condition prior to treatment in the subject is determined;

an effective amount of ethyl icosapentate administering to the subject in combination with at least one drug selected from the group consisting of lipid-lowering drugs, HMG-CoA reductase inhibitors (stains), fibrates, probucol, ezetimibe, ursodiol (UDCA), taurine, betaine, N-acetylcysteine, s-adenosylmethionine (SAM-e), milk thistle, anti-TNF therapies, probiotics, anti-diabetic medications: biguanides (metformin), insulin, sulfonylureas, alpha-glucosidase inhibitors (acarbose), dipeptidyl-peptidase 4 inhibitors (sitagliptin, saxagliptin, alogliptin, vildagliptin, linagliptin, etc.), phenylalanine derivatives (nateglinide, repaglinide), anti-platelet therapy, anti-thrombotic agents, Glucagon-like peptide-1 (GLP-1) receptor agonists (liraglutide, exenatide, taspoglutide, etc.), PDE-4 inhibitor, angiotensin II-1 type receptor antagonist (ARB: losartan, etc.), polyenephosphatidylcholine, antioxidant (vitamine E, vitamin C, nicotinic acid tocopherol, etc.), and pentoxifylline; and

the NAS score in the subject is improved (i) to a composite score of 3 or less than 3, or (ii) by 2 or more than 2 across at least two of the NAS components, together with no worsening of the fibrosis stage score, optionally improving at least one of items mentioned in Tables 1 and 2.

In a further embodiment of the invention, ethyl icosapentate for use in the treatment or alleviation of symptoms of NASH wherein a subject an effective amount of ethyl icosapentate is administered, the subject has NASH characterized by baseline levels of ALT of between 5 to 300 U/L and at least one criteria selected from the group consisting of a NAS score of 4 or more than 4, a steatosis score of 1 or more than 1, lobular inflammation score of 1 or more than 1 and either (i) a fibrosis stage of at least 1a or (ii) ballooning; and the NAS score in the subject (i) to a composite score of 3 or less than 3, or (ii) by 2 or more than 2 across at least two of the NAS components, together with no worsening of the fibrosis stage score is improved.

In another embodiment of the invention, ethyl icosapentate for use in reducing steatosis, liver lobular inflammation, ballooning and/or liver fibrosis in a subject in need thereof, wherein, an effective amount of ethyl icosapentate (EPA-E) is administered to a subject; at least one condition selected from the group consisting of the steatosis, lobular inflammation, ballooning and liver fibrosis condition of said subject is improved, and no worsening of said fibrosis stage score; and said subject exhibits the following changes in said at least one marker as compared to a baseline pretreatment level of at least 1% reduction for ALT, AST, Triglycerides (TG), TG/HDL-C ratio, Free fatty acid, Arachidonic acid (AA), monounsaturated fatty acid (MUFA), Palmitoleic acid, Oleic acid, Oleic Acid/Stearic acid ratio, Palmitoleic acid/Palmitic acid ratio, Stearic acid/Palmitic acid ratio, gamma-linolenic acid/Linolenic acid ratio, Adrenic acid/AA ratio, Ferritin, Thioredoxin, Tumor necrosis factor-alpha (TNF-alpha), sTNF-R1 (Tumor necrosis factor receptor I, soluble), sTNF-R2 (Tumor necrosis factor receptor II, soluble), Hs-CRP, CTGF, sCD40, Leptin, complement factor D, CK18 fragment, serum HMGB1, soluble Fas antigen, Hyaluronic acid, Type IV collagen (7s domain), procollagen III peptide or PAI-1; at least 5% increase for EPA or EPA/AA ratio; at least 1% increase for DPA, AA/Homo-gamma-linolenic acid ratio or Serum adiponectin; no worsening of ALP, bilirubin, GGT, Albumin, HDL-C, LDL-C, Total Cholesterol (TC), non-HDL-C, HOMA-IR, HbAlc, Fasting plasma glucose, postprandial plasma glucose, OGTT, platelet count or BMI.

In another embodiment of the invention, the ethyl icosapentate for use in reducing steatosis, liver lobular inflammation, ballooning and/or liver fibrosis in a subject in need thereof, wherein;

a baseline level in blood or physical condition prior to treatment in the subject having at least one item or any combination of two or more items selected from the pretreatment baseline of the items mentioned in Tables 1 and 2 is determined;

an effective amount of ethyl icosapentate (EPA-E) is administered to the subject;

at least one condition selected from the group consisting of the steatosis, lobular inflammation, ballooning and liver fibrosis condition of said subject without worsening said fibrosis stage score is improved; and

said subject exhibits the described changes in at least one of items mentioned in Tables 1 and 2 as compared to a baseline pre-treatment level of the item.

In another embodiment of the invention, the ethyl icosapentate for use in treatment or alleviation of symptoms NASH, wherein the subject is taking at least one drug selected from the group consisting of lipid-lowering drugs, HMG-CoA reductase inhibitors (stains), fibrates, probucol, ezetimibe, ursodiol (UDCA), taurine, betaine, N-acetylcysteine, s-adenosylmethionine (SAM-e), milk thistle, anti-TNF therapies, probiotics, anti-diabetic medications: biguanides (metformin), insulin, sulfonylureas, alpha-glucosidase inhibitors (acarbose), dipeptidyl-peptidase 4 inhibitors (sitagliptin, saxagliptin, alogliptin, vildagliptin, linagliptin, etc.), phenylalanine derivatives (nateglinide, repaglinide), anti-platelet therapy, anti-thrombotic agents, Glucagon-like peptide-1 (GLP-1) receptor agonists (liraglutide, exenatide, taspoglutide, etc.), PDE-4 inhibitor, angiotensin II-1 type receptor antagonist (ARB: losartan, etc.), polyenephosphatidylcholine, antioxidant (vitamine E, vitamin C, nicotinic acid tocopherol, etc.), and pentoxifylline.

In another embodiment of the invention, ethyl icosapentate for use in the treatment or alleviation of symptoms of NASH wherein an effective amount of ethyl icosapentate is administered to a subject, wherein the subject is possible or definite NASH, and a baseline level in blood or physical condition prior to treatment in the subject of at least one member selected from the group consisting of ALT, AST, AST/ALT ratio, ALP, bilirubin, GGT, Albumin, HDL-C, LDL-C, TG, TC, TG/HDL-C ratio, non-HDL-C, Free fatty acid, AA, EPA, DPA, DHA, EPA/AA ratio, DPA/AA ratio, DHA/AA ratio, DHA/DPA ratio, MUFA, Palmitoleic acid, Oleic acid, Oleic acid/Stearic acid ratio, Palmitoleic acid/Palmitic acid ratio, Stearic acid/Palmitic acid ratio, gamma-linolenic acid/Linolenic acid ratio, AA/Homo-gamma-linolenic acid ratio, Adrenic acid/AA ratio, Ferritin, Thioredoxin, TNF-alpha, sTNF-R1, sTNF-R2, Hs-CRP, CTGF, sCD40, HOMA-IR, HbAlc, Glucose, Fasting plasma glucose, postprandial plasma glucose, OGTT, Leptin, Serum adiponectin, complement factor D, CK18 fragment, serum HMGB1, soluble Fas antigen, Hyaluronic acid, Type IV collagen (7s domain), procollagen III peptide, PAI-1, platelet count or BMI is determined.

In another embodiment of the invention, the ethyl icosapentate for use in the treatment or alleviation of symptoms of NASH, wherein the subject is possible or definite NASH and an effective amount of ethyl icosapentate is administered to a subject, wherein a baseline level in blood or physical condition prior to treatment in the subject of at least one item or any combination of two or more items selected from the items mentioned in Tables 1 and 2 is determined.

In another embodiment of the invention, the ethyl icosapentate for use in treatment or alleviation of symptoms NASH, wherein the subject is possible or definite NASH and the subject is taking at least one drug selected from the group consisting of lipid-lowering drugs, HMG-CoA reductase inhibitors (stains), fibrates, probucol, ezetimibe, ursodiol (UDCA), taurine, betaine, N-acetylcysteine, s-adenosylmethionine (SAM-e), milk thistle, anti-TNF therapies, probiotics, anti-diabetic medications: biguanides (metformin), insulin, sulfonylureas, alpha-glucosidase inhibitors (acarbose), dipeptidyl-peptidase 4 inhibitors (sitagliptin, saxagliptin, alogliptin, vildagliptin, linagliptin, etc.), phenylalanine derivatives (nateglinide, repaglinide), anti-platelet therapy, anti-thrombotic agents, Glucagon-like peptide-1 (GLP-1) receptor agonists (liraglutide, exenatide, taspoglutide, etc.), PDE-4 inhibitor, angiotensin II-1 type receptor antagonist (ARB: losartan, etc.), polyenephosphatidylcholine, antioxidant (vitamine E, vitamin C, nicotinic acid tocopherol, etc.), and pentoxifylline.

In another embodiment of the invention, the ethyl icosapentate for use in the treatment or alleviation of symptoms of NASH wherein an effective amount of ethyl icosapentate is administered to a subject, wherein the subject is possible or definite NASH, and exhibits the following changes in said at least one marker as compared to a baseline pre-treatment level of at least 1% reduction for ALT, AST, TG, TG/HDL-C ratio, Free Fatty acid, AA, MUFA, Palmitoleic acid, Oleic acid, Oleic acid/Stearic acid ratio, Palmitoleic acid/Palmitic acid ratio, Stearic acid/Palmitic acid ratio, gamma-linolenic acid/Linolenic acid ratio, Adrenic acid/AA ratio, Ferritin, Thioredoxin, TNF-alpha, sTNF-R1, sTNF-R2, Hs-CRP, CTGF, sCD40, Leptin, complement factor D, CK18 fragment, serum HMGB1, soluble Fas antigen, Hyaluronic acid, Type IV collagen (7s domain), procollagen III peptide or PAI-1; at least 5% increase for EPA or EPA/AA ratio; at least 1% increase for DPA, AA/Homo-gamma-linolenic acid ratio or Serum adiponectin; no worsening of ALP, bilirubin, GGT, Albumin, HDL-C, LDL-C, TC, non-HDL-C, HOMA-IR, HbAlc, Glucose, Fasting plasma glucose, postprandial plasma glucose, OGTT, platelet count or BMI.

In another embodiment of the invention, the ethyl icosapentate for use in the treatment or alleviation of symptoms of NASH wherein an effective amount of ethyl icosapentate is administered to a subject being possible or definite NASH, and exhibits the described changes of after dosing value in said at least one item selected from the items mentioned in Tables 1 and 2 as compared to a baseline pre-treatment level thereof.

In another embodiment of the invention, the ethyl icosapentate for use in the treatment or alleviation of symptoms of NASH wherein an effective amount of ethyl icosapentate is administered to a subject, wherein the subject is taking at least one drug selected from the group consisting of lipid-lowering drugs, HMG-CoA reductase inhibitors (stains), fibrates, probucol, ezetimibe, ursodiol (UDCA), taurine, betaine, N-acetylcysteine, s-adenosylmethionine (SAM-e), milk thistle, anti-TNF therapies, probiotics, anti-diabetic medications: biguanides (metformin), insulin, sulfonylureas, alpha-glucosidase inhibitors (acarbose), dipeptidyl-peptidase 4 inhibitors (sitagliptin, saxagliptin, alogliptin, vildagliptin, linagliptin, etc.), phenylalanine derivatives (nateglinide, repaglinide), anti-platelet therapy, anti-thrombotic agents, Glucagon-like peptide-1 (GLP-1) receptor agonists (liraglutide, exenatide, taspoglutide, etc.), PDE-4 inhibitor, angiotensin II-1 type receptor antagonist (ARB: losartan, etc.), polyenephosphatidylcholine, antioxidant (vitamine E, vitamin C, nicotinic acid tocopherol, etc.), and pentoxifylline.

In another embodiment of the invention, the ethyl icosapentate for use in the treatment or alleviation of symptoms of NASH wherein an effective amount of ethyl icosapentate is administered to a subject, wherein the subject is taking an HMG-CoA reductase inhibitor (statins; pravastatin sodium, simvastatin, pitavastatin calcium, atorvastatin calcium hydrate, rosuvastatin calcium, etc.).

In another embodiment of the invention, the ethyl icosapentate for use in the treatment or alleviation of symptoms of NASH wherein an effective amount of ethyl icosapentate is administered to a subject, wherein the subject is taking a Glucagon-like peptide-1 (GLP-1) receptor agonist (liraglutide, exenatide, taspoglutide, etc.).

In another embodiment of the invention, the ethyl icosapentate for use in the treatment or alleviation of symptoms of NASH wherein an effective amount of ethyl icosapentate is administered to a subject in combination with at least one drug selected from the group consisting of lipid-lowering drugs, HMG-CoA reductase inhibitors (stains), fibrates, probucol, ezetimibe, ursodiol (UDCA), taurine, betaine, N-acetylcysteine, s-adenosylmethionine (SAM-e), milk thistle, anti-TNF therapies, probiotics, anti-diabetic medications: biguanides (metformin), insulin, sulfonylureas, alpha-glucosidase inhibitors (acarbose), dipeptidyl-peptidase 4 inhibitors (sitagliptin, saxagliptin, alogliptin, vildagliptin, linagliptin, etc.), phenylalanine derivatives (nateglinide, repaglinide), anti-platelet therapy, anti-thrombotic agents, Glucagon-like peptide-1 (GLP-1) receptor agonists (liraglutide, exenatide, taspoglutide, etc.), PDE-4 inhibitor, angiotensin II-1 type receptor antagonist (ARB: losartan, etc.), polyenephosphatidylcholine, antioxidant (vitamine E, vitamin C, nicotinic acid tocopherol, etc.), and pentoxifylline.

In another embodiment of the invention, the ethyl icosapentate for use in reducing at least one marker as compared to a baseline pre-treatment level of Hs-CRP, CTGF, sCD40, Leptin, complement factor D, serum HMGB1, soluble Fas antigen or procollagen III peptide in a subject, comprising administering to a subject an effective amount of ethyl icosapentate (EPA-E), wherein the subject has NASH.

In another embodiment of the invention, the ethyl icosapentate for use in determining efficacy of NASH treatment by (i) administering to a subject an effective amount of EPA-E, (ii) measuring at least one marker of the items mentioned in Tables 1 and 2 during the treatment, (iii) comparing the measured levels of markers to established levels in advance, and optionally (iv) determining whether the treatment is efficacious.

DETAILED DESCRIPTION OF THE INVENTION

The compositions and methods of the present invention are useful for the treatment of NASH by administration of an effective amount of ethyl icosapentate.

Icosapentaenoic acid (EPA) is a known omega-3 polyunsaturated, long-chain fatty acid. Omega-3 fatty acids are known as components of oils, such as fish oil, and a variety of commercial products are promoted as containing omega-3 fatty acids, or their esters, derivatives, conjugates and the like. Icosapentaenoic acid (EPA) is also per se known in its ethyl ester form, ethyl icosapentate (EPA-E). According to the present invention, EPA-E can be administered in a composition. EPA-E content in the total fatty acid of the compositions of the present invention are not particularly limited as long as the composition contains EPA-E as its effective component and intended effects of the present invention are attained, high purity EPA-E is preferably used; for example, the composition having a proportion of the EPA-E of preferably 40% by weight or more, more preferably 90% by weight or more, and still more preferably 96.5% by weight or more in total of the fatty acids and their derivatives. EPA-E can be administered to patients in a highly purified form, including the product known as Epadel (Trade mark) (Mochida Pharmaceutical Co., Ltd., Tokyo Japan). The compositions of EPA-E are administered according to the invention to a subject or patient to provide the patient with a dosage of about 0.3-10 g per day of EPA-E, alternatively 0.6-6 g per day, alternatively 0.9-3.6 g per day or specifically about 300-4000 mg per day or preferably 900-3600 mg per day or more preferably about 1800-2700 mg per day of EPA-E. The compositions of EPA-E are administered according to the invention to a subject or patient preferably one two, or three times per day.

Since EPAs are highly unsaturated, the preparation as described above preferably contains an antioxidant at an amount effective for suppressing oxidation of the EPAs. Exemplary antioxidants include butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, gallic acid, pharmaceutically acceptable quinone, and alpha-tocopherol.

The composition to be administered can contain other fatty acids, especially any omega-3 unsaturated fatty acid, especially DHA-E. The ratio of EPA-E/DHA-E in the composition, the content of EPA-E and DHA-E in the total fatty acids and administration amount of EPA-E and DHA-E are not limited but the ratio is preferably 0.8 or more, more preferably 1.0 or more, still more preferably 1.2 or more. The composition is preferably highly purified; for example, the proportion of EPA-E+DHA-E in the fatty acids and their derivatives is preferably 40% by weight or more, more preferably 80% by weight or more, and still more preferably 90% or more. The daily amount in terms of EPA-E+DHA-E is typically 0.3 to 10.0 g/day, preferably 0.5 to 6.0 g/day, and still more preferably 1.0 to 4.0 g/day. The low content of other long chain saturated fatty acids is preferred, and among the long chain unsaturated fatty acids, the content of omega-6 fatty acids, and in particular, the content of arachidonic acid is preferably as low as less than 2% by weight, and more preferably less than 1% by weight. For example, soft capsule (Lovaza) (Trade mark) or Omacor (Trade mark) containing about 46% by weight of EPA-E and about 38% by weight of DHA-E is commercially available in the U.S., EP and other countries as a therapeutic agent for hyerptriglyceridemia.

Patients treated for NASH can be administered EPA-E according to the invention for 3, 6 or 9 months, or for 1 year or more and can be administered EPA-E in one, two or three dosage per day, or other multiple doses per day including 1 to about 10, 1 to 8, 1 to 6, 1 to 4 or 1 to 2 dosage units per day as appropriate for patient therapy. The term “dose unit” and “dosage unit” herein refer to a portion of a pharmaceutical composition that contains an amount of EPA-E for a single administration to a subject.

While meal affects absorption of the EPA-E, and the administration of the EPA-E is preferably conducted during the meal or after the meal, and more preferably immediately after the meal (within 30 minutes after the meal). The self-emulsifying composition has excellent absorption under fasting, and therefore, it exhibits the intended effects even when administered at a timing other than during, after, or immediately after the meal.

Compositions comprising EPA-E useful for the invention include commercially available compositions of EPA-E, such as Epadel (Trade mark) noted above. Compositions comprising EPA-E may be administered in tablet, capsule, microcapsule, jelly, enteric preparation, extended release preparation, powder or any other solid oral dosage form, as a liquid, emulsion, self-emulsifying composition, as a soft gel capsule or other capsule form, or other appropriate and convenient dosage forms for administration to a patient in need thereof. Compositions can also include pharmaceutically acceptable excipients known to those of ordinary skill in the art including surfactants, oils, co-solvents or combinations of such excipients, together with stabilizers, emulsifiers, preservatives, solubilizers and/or other non-active pharmaceutical ingredients known to those of skill in the art relative to the preparation of pharmaceutical compositions.

1. Evaluation Criteria for Patients

As noted above, the “gold-standard” for a complete diagnosis of NASH involves a liver biopsy. Patients or subjects treated for NASH according to the present invention can also be evaluated for the following criteria, including evaluation prior to initiation of treatment in order to provide a baseline level or score for the criteria as well as evaluation after the dosing regimen to evaluate any improvement in the criteria.

a. NAS Score:

A non-alcoholic fatty liver disease activity score (NAS) is defined as the unweighted sum of the values for steatosis (ranging from 0-3), lobular inflammation (ranging from 0-3) and ballooning (ranging from 0-2), thereby providing a range of NAS score of from 0 to 8. (See Kleinen et al., Design and Validation of a Histological Scoring System for Nonalcoholic Fatty Liver Disease, Hepatology, Vol. 41, No. 6, 2005, pp. 1313-1321) Patients treated for NASH according to the present invention can show a NAS score prior to treatment of 4 or more than 4, with a minimum score of 1 each for steatosis and lobular inflammation plus either ballooning or at least 1a sinusoidal fibrosis and a finding of possible or definite steatohepatitis. After dosing/treatment, such as for one year, patients can show a composite NAS score of 3 or less than 3, 2 or less than 2, or 1 or less than 1, together with no worsening in fibrosis. Alternatively, patients can show an improvement in NAS by a value of 2 or more than 2 across at least two of the NAS components, together with no worsening in fibrosis. Alternatively, patients can show an improvement in NAS score by 3 or more than 3, 4 or more than 4, 5 or more than 5, 6 or more than 6, 7 or more than 7, or 8 or more than 8.

b. Steatosis:

Steatosis is broadly understood to describe a process involving the abnormal retention of lipids within the liver, which accumulation inhibits the normal liver functions. Liver biopsy enables analysis and scoring of steatosis in a patient, with scores ranging from 0-3. Patients treated for NASH according to the present invention can have a steatosis score of 1, 2 or 3, such as between about 2 and about 3. After treatment, it is desired for patients to exhibit no worsening of steatosis, alternatively a reduction of at least 1 in the steatosis score, or a reduction of 2 or 3 in the steatosis score. Steatosis is traditionally graded with a score of 1 indicating the presence of fat droplets in less than 33% of hepatocytes, a score of 2 indicating fat droplets observed in 33-66% of hepatocytes, and a score of 3 indicating observation of fat droplets in greater than 66% of hepato sites. (See Kleinen et al., Design and Validation of a Histological Scoring System for Nonalcoholic Fatty Liver Disease, Hepatology, Vol. 41, No. 6, 2005, pp. 1313-1321)

c. Lobular Inflammation:

Lobular inflammation is also evaluated upon liver biopsy and scored with values of 0-3. (See Kleinen et al., Design and Validation of a Histological Scoring System for Nonalcoholic Fatty Liver Disease, Hepatology, Vol. 41, No. 6, 2005, pp. 1313-1321 Table 1) Patients to be treated for NASH can have lobular inflammation scores of 1, 2 or 3, alternatively ranging between 1 and 2 or 2 and 3. After treatment, patients can have a reduction in lobular inflammation score of at least 1, alternatively a reduction of 2 or 3 in lobular inflammation score, and at least no worsening of the lobular inflammation score.

d. Ballooning:

Ballooning of hepatocytes is generally scored with values of 0-2, (See Kleinen et al., Design and Validation of a Histological Scoring System for Nonalcoholic Fatty Liver Disease, Hepatology, Vol. 41, No. 6, 2005, pp. 1313-1321 Table 1), and patients treated for NASH according to the present invention can have ballooning scores of 0-2, including specific values of 1 or 2, and alternatively a score ranging from 1 to 2. After treatment, patients can show at least no worsening of the ballooning score, alternatively a reduction of at least one value lower in the ballooning score, and alternatively a reduction of two in the value of the ballooning score.

e. Fibrosis Stage

Fibrosis is also evaluated upon liver biopsy and scored with values of 0-4, the scores being defined as: 0 represents no fibrosis, 1 represents perisinusoidal or periportal fibrosis, 1a represents mild, zone 3, perisinusoidal fibrosis; 1b represents moderate zone 3, perisinusoidal fibrosis; 1c represents portal/periportal fibrosis; 2 represents perisinusoidal and portal/periportal fibrosis; 3 represents bridging fibrosis; and 4 represents cirrhosis. (See Kleinen et al., Design and Validation of a Histological Scoring System for Nonalcoholic Fatty Liver Disease, Hepatology, Vol. 41, No. 6, 2005, pp. 1313-1321) Patients treated according to the present invention can have a fibrosis stage score of 0-3, including 0, 1, 1a, 1b, 1c, 2 or 3, and can have a fibrosis stage score of at least 1a. After treatment, patients can have a fibrosis stage score that is at least no worse than the baseline score, and alternatively can have a reduction in the fibrosis stage score of at least one level, alternatively at least two or three levels.

2. Additional Criteria/Markers for Evaluation of Patients

As noted above, while liver biopsy is considered the “gold-standard” for clinical assessment of NASH, the condition can also be accompanied or associated with abnormal levels of liver enzymes and other biological blood components. Therefore, patients treated for NASH according to the present invention can also be evaluated for baseline scores of the following criteria before treatment, and evaluated after treatment for possible changes in those criteria. The evaluated criteria can comprise one or more of the following criteria set forth in Tables 1 and 2.

In the present invention, a biological sample of the patient is collected and used to obtain measurement values. Specific examples of the biological sample include blood, plasma, serum, urine, body fluids, and tissues, but are not limited thereto. The biological sample is preferably blood, plasma or serum. The biological sample is collected from a subject by a known method.

In the present invention, a normal value is measured in accordance with a known measuring method if the normal value is known as one of the blood test indices used to detect NASH, or in accordance with a measuring method following a reference document or the like if a common measuring method for the normal value is not established.

For instance, the normal values shown in Tables 1 and 2, except BMI, can be each measured with a biological sample of either blood, plasma or serum. Fatty acids in blood may be used to measure fatty acids. Table 3 shows a list of some reference documents which recite the particulars of the measurement method.

Unless otherwise specified, the fatty acid amount and the fatty acid composition ratio as used in the present invention may be the amount and the composition ratio of fatty acids in any of the plasma, serum and liver. It is also possible indeed to use the fatty acid amount and the fatty acid composition ratio in a specified fraction, such as LDL or VLDL in the blood. It, however, is desirable to use the amount and the composition ratio of fatty acids in the plasma or the serum because of the simplicity of measurement. Each fatty acid to be employed for the calculation of the fatty acid amount and the fatty acid composition ratio is not particularly limited in unit of amount, that is to say, its amount may be expressed in mole, mole percent, a unit of weight, percent by weight, or the like. The sole unit, and the sole method of calculating fatty acid amount and the fatty acid composition ratios should be used if the evaluation is to be made by the comparison of the fatty acid amount and the fatty acid composition ratio over time. It is particularly desirable to calculate the fatty acid amount and the fatty acid composition ratio from fatty acid amounts expressed in mole percent of the total amount of fatty acids. The weight/volume concentration (e.g., micro g/ml), the mole/volume concentration (e.g., mol/L) or the like may also be used for the calculation.

In this description, the term “plasma fatty acid” refers to a plasma total fatty acid unless otherwise specified. It is also possible to use a plasma free fatty acid for the inventive index for the evaluation of the subject's condition or therapeutic effects. The term “liver fatty acid” refers to a liver total fatty acid unless otherwise specified. A liver free fatty acid may optionally be used.

The fatty acid composition may be determined by any method practicable by a person of ordinary skill in the art of the present invention, while it is particularly preferable to determine the composition according to a usual manner.

TABLE 1

Pre-treatment baseline
After dosing (effect) values

Item (Typical

Observable

Observable

Normal
Typical
Ranges or

Ranges or

Values, Units)
Range(s)
Values
Typical Range(s)
Values

ALT (alanine
10-300
Lower limit
at least 1%
1 to about 95%

aminotransferase,

range values of
lower
reduction

GPT)

10, 50, 100, 150,

(6-41 U/L)

or 200, upper

limit range

values of 100,

150, 200, 250,

or 300, ranges

of 10-300, 10-200,

10-150, 10-100,

100-200,

2000-3000

AST (asparate
10-250
Lower limit
at least 1%
1 to about 95%

aminotransferase,

range values of
lower
reduction

GOT)

10, 50, 100, 150,

(9-34 U/L)

or 200, upper

limit range

values of 100,

150, 200, 250,

or 300, ranges

of 10-300, 10-200,

10-150, 10-100,

100-200,

200-300

AST/ALT ratio

upper limit

range values of

0.5, 0.7, 0.8, 1,

1.2, 2; ranges of

0.5-2, 0.5-1, 1-2

alkaline
80-300
ranges of 50-600
no worsening
no worsening, 1

phospatase

to about 90%

(ALP)

reduction,

(80-260 IU/L)

300 IU/L or less,

250 IU/L or less

Total bilirubin
High

no worsening
no worsening, 1

(0.2-1.2 mg/dL)
compared to

to about 90%

average level

reduction,

of normal

subject

Gamma-
High

no worsening
no worsening, 1

Glutamyl
compared to

to about 90%

Transferase
average level

reduction,

(GGT or γGTP)
of normal

100 U/L or less,

(males: 5-60 U/L)
subject

70 U/L or less

Albumin (3.8-5.2 g/dl)
Low

no worsening
no worsening, 1

compared to

to about 90%

average level

increase, ranges

of normal

of 3-6 g/dl, 3.5-5.5 g/dl

subjects

HDL-C (high
less than 55
less than
no worsening, at
no change, 1-90%

density

60 mg/dl, 55, 50,
least 1%
increase,

lipoprotein

45, 40, 35, 30,
increase
40 mg/dl or

cholesterol)

25, or 25 mg/dl;

more

(35-60-mg/dl)

ranges of 25-55,

30-40 mg/dl, 40-50 mg/dl,

50-60 mg/dl,

at

least 60

LDL-C (low
100-200
at least 70 mg/dl,
no worsening
no change, 1-90%

density

100,

reduction

lipoprotein

120, 130 140

less than

cholesterol)

150, 170, 190,

160 mg/dl, 140,

(50-130 mg/dl)

or 200 or a

130, 120, 100,

range of 70-300,

70 mg/dl

70-250, 70-200,

100-250, 100-200,

130-200,

140-180, 100-130,

130-160,

160-190

Triglycerides
100-1000
at least 80 mg/dl,
at least 1%
1 to about 90%

(TG) (fed or

100, 150,
lower
reduction, 500 mg/dl

fasting, 50-150 mg/dl)

180, 200, 300,

or less,

500, 700, 1000,

300, 200, 150,

1200, or 1500,

100 mg/dl or

or less than 150,

less

or a range of

100-2500, 100-1500,

100-1000,

150-500, 200-500,

150-300,

150-200, 200-500

Total
170-300
a range of 130-300 mg/dl,
no worsening
no change, 1-90%

Cholesterol

200-220,

reduction

(TC) (100-200 mg/dl)

220-240,

240-260, or at

least 260, or less

than 200 mg/dl

TG and HDL-C
High TG and
TG: at least 150,
no worsening

low HDL-C
200, 500 mg/dl

(ex. TG ≧150 mg/dl
HDL-C; less than

and
40, 50 mg/dl

HDL≦40 mg/dl

TG/HDL-C
at least 3.75
at least 2, 2.5, 3,
at least 1%
no worsening, at

ratio

3.75, 4, 5, 10, or
lower
least 1% lower, or

ranges of 2-3.75,

1-90% reduction

3.75-10

Non-HDL-C
at least 130
at least
no worsening
no worsening,

(mg/dl)

100 mg/dl, 130,

or at least 1%

150, 160, 170,

lower, or less

190, a range of

than 130 mg/dl,

100 to 250

150, 160, 170,

190

Free fatty acid
at least 400
less than 400, at
at least 1%
no change, or at

(μEq/l)

least 400, 600,
lower
least 1 to 90%

(140-850)

800, 1000

reduction

Eicosapentaenoic
less than 0.5/
less than 1,
at least 5%
5 to about 200%

Acid/Arachidonic
low
0.75, 0.5, 0.1,
increase
increase, about

Acid
compared to
ranges of 0.01-2

2-200-fold

(EPA/AA)
average level

increase

(ex. (mol/%)/
or normal

(mol/%)
subjects

Arachidonic
High

at least 1%
no change, 1 to

Acid (AA)
compared to

lower
about 90%

(ex. mol/%)
average level

reduction

of normal

subjects

Eicosapentaenoic
low compared

at least 5%
5 to about 200%

Acid (EPA)
to average

increase
increase, about

(ex. mol/%)
level of

2-500-fold

normal

increase

subjects

Docosapentaenoic
low compared

at least 1%
1 to about 95%

Acid
to average

increase
increase

(DPA)
level of

(ex. mol/%)
normal

subjects

Docosahexaenoic
low compared

Acid (DHA)
to average

(ex. mol/%)
level of

normal

subjects

DPA/AA ratio
low compared

to average

level of

normal

subjects

DPA/AA ratio
low compared

to average

level of

normal

subjects

DHA/DPA
low compared

ratio
to average

level of

normal

subjects

Monounsaturated
High

at least 1%
no change, at

fatty acid
compared to

lower
least 1% lower

(MUFA)
average level

(ex. mol/%)
of normal

subjects

Palmitoleic
High

at least 1%
no change, at

acid (16:1 n7)
compared to

lower
least 1% lower

(ex. mol/%)
average level

of normal

subjects

Oleic acid
High

at least 1%
no change, a

(18:1 n9) (ex.
compared to

lower
least 1% lower

mol/%)
average level

of normal

subjects

Oleic acid
High

at least 1%
no change, at

(18:1 n9)/
compared to

lower
least 1% lower

stearic acid
average level

(18:0) ratio
of normal

subjects

Palmitoleic
High

at least 1%
no change, at

acid (16:1)/
compared to

lower
least 1% lower

Palmitic acid
average level

(16:0) ratio
of normal

subjects

Stearic acid
High

no change, or at
no change, or at

(18:0)/
compared to

least 1% lower
least 1% lower

Palmitic acid
average level

(16:0) ratio
or normal

subjects

γ-linolenic
High

no change, or at
no change, or at

acid(18:3 n6)/
compared to

least 1% lower
least 1% lower

Linolenic acid
average level

(18:2 n6) ratio
subjects

AA/Homo-γ-
low compared

no change, or at
no change, or at

linolenic acid
to average

least 1%
least 1%

(20:3 n6) ratio
level of

increase
increase

normal

subjects

Acrenic acid
High

no change, or at
no change, or at

(22:4 n6)/
compared to

least 1% lower
least 1% lower

AA ratio
average level

of normal

subjects

Ferritin

at least 100,
at least 1%
at least 1 to

(ng/mL)

120, 150, 200,
lower
about 95%

250, 300, 350,

lower

400, or 500

Thioredoxin

at least 15, 20,
at least 1%
at least 1 to

(ng/mL)

25, 30, 35, 40,
lower
about 95%

45, or 50

lower

TNFα (Tumor
at least 1.5
at least 1, 1.5,
at least 1%
at least 1 to

necrosis

1.6, 1.7, 1.79,
lower
about 95%

factor-

1.8, 1.9, 2.0, 2.2,

lower

α)(pg/mL)

2.5, 3, 3.5, 4, 5,

(1.79 or less)

6, 7 or 10

sTNF-R1

at least 400,
at least 1%
at least 1 to

(Tumor

500, 600, 700,
lower
about 95%

necrosis factor

800, 900, 1000,

lower

receptor I,

1100, 1200,

soluble)

1500, or 2000

(pg/mL)

sTNF-R2

at least 500,
at least 1%
at least 1 to

(Tumor

700, 1000, 1200,
lower
about 95%

necrosis factor

1500, 1700,

lower

receptor

2000, 2200,

II, soluble)

2500, 2700, or

(pg/mL)

3000

High
0.2
0.1 or more, 0.2,
at least 1%
at least 5 to

Sensitivity C-

0.3, 0.4, 0.5 or
lower
about 95%

reactive

more, ranges of

lower

protien (Hs-

0.1-1, 0.1-0.8,

CRP, mg/dl)

0.1-0.5, 0.2-0.5

Connective
High

at least 1%
at least 5 to

Tissue Growth
compared to

lower
about 95%

Factor (CTGF)
average level

lower

of normal

subject

Serum Soluble

5 pg/ml or
at least 1%
at least 5 to

CD40 (sCD40,

more, 10, 20,
lower
about 95%

pg/ml)

30, 50, 70, 100,

lower

120, 150, 170,

200, 220, 250,

300, 350, 400,

450, 500 or

more

Insulin
1.5 or more
1.6 or less/1.5
no worsening
no change, at

resistance

or more, 1.6, 2,

least 1 to about

Index (HOMA-

2.5, 3, 3.5, 4

50% lower

IR) (1.6 or

less)

Glycated
5.7 or more
a range of 4.3-5.8,
no worsening
no change, at

hemoglobin

5.7-6.4, 5.8-6.5,

least 1 to about

(HbA1c) (4.3-5.8%)

6.5-7.0, 7.0-8.0/

50% lower

5.7 or

more, 5.8, 6,

6.5, 7, 7.5, 8, or

8.5

Fasting
100 or more
less than 100/
no worsening
no change, or at

plasma

100 or more,

least 1 to about

glucose (FPG)

110, 120, 126,

50% lower

(mg/dl)

130, 150, 200,

(less than 100)

250, 300/

ranges of 100-110,

100-126

Postprandial
140 or more
less than 140,
no worsening
no change, or at

plasma

160, 200/

least 1 to about

glucose (after

140 or more,

50% lower

a meal)

170, 180, 200,

250, 300, 350

400/ranges of

140-200, 140-170,

170-200

two-hour
140-200
less than 140,
no worsening
no change, or at

glucose levels

160, 200/140 or

least 1 to about

on the 75-g

more, 170, 180,

50% lower

oral glucose

200, 250, 300,

tolerance test

350, 400/

(mg/dl)

ranges of 140-200,

(OGTT)

140-170,

170-200

Leptin (ng/ml)

5 ng/ml or
at least 1% lower
at least 1 to

more, 10, 12,

about 95%

15, 17, 20,

reduction

22, 25, 30, 35,

40 or more

Serum

5 μg/mL or less,
at least 1%
no change, at

adiponectin

4.5, 4, 3.5, or 3 μg/mL
increase
least 1 to about

(μg/mL)

or less

95% increase

complement
High

at least
at least 1 to

factor D
compared to

15% lower
about 95%

average level

reduction

of normal

subject

CK18
High

at least 1% lower
at least 1 to

fragment
compared to

about 95%

average level

reduction

of normal

subject

serum High
High

at least 1% lower
at least 1 to

mobility group
compared to

about 95%

box 1 protein
average level

reduction

(HMGB1)
of normal

subject

soluble Fas
High

at least 1% lower
at least 1 to

antigen
compared to

about 95%

(CD95, sFas)
average level

reduction

of normal

subject

Hyaluronic

25 ng/mL or
at least 1%
at least 1 to

acid

more, 50, 70,
lower
about 95%

(50 ng/mL or

100, 120, 150,

reduction

less)

200, 250, or 300

or more; 200 mL

or less, 100, 70,

or 50 or less

Type IV

5 ng/mL or
at least 1%
at least 1 to

collagen (7s

more, 6, 7, 8,
lower
about 95%

domain)

10, 12, 15, or 20

reduction

(6 ng/mL or

or more;

less)

25 ng/mL or

less, 20, 15, 10,

or 6 or less

procollagen III

0.2 U/ml or
at least 1%
at least 1 to

peptide 0.3-0.8 U/ml

more, 0.3, 0.5,
lower
about 95%

0.7, 1, 1.2, 1.5,

reduction

2, 2.5, 3, 3.5, or

4 or more; 10 or

less, 8, 5, 3, 1, or

0.8 or less

PAI-1 (ng/mL)
50 or more

50 or less

platelet count
150000-300000
400000/μL or
no change
no change, at

150000-400000/

less, 300000,

least 1%

μL

200000/a range

increase

of 150000-300000

BMI
18.5-40
18.5 or more,
no change
no change, at

20, 25, 30, 35,

least 1%

40, or 50 or

reduction

more;/50 or

less, 40, 30, 25,

20 or 18.5 or

less; or range of

18.5-25, 25-30,

30-35, 35-40

Direct Bilirubin
High compared

No worsening
No worsening,

(0-0.4 mg/dl)
to average level

1 to about 90%

of normal

reduction

subject

Oleic acid (C18:1
High compared

At least 1% lower
No change, at

n9)/Palmitic
to average level

least 1% lower

acid
of normal

(C16:0)ratio
subject

EPA/AA ratio
Low EPA/AA
EPA/AA ratio

EPA/AA ratio

and Hs-CRP
ratio and high
being 1.0 or less,

increases; Hs-CRP

Hs-CRP
0.75, 0.6, 0.5, 0.4,

decreases

0.25 or less; Hs-

CRP being

0.1 mg/dl or

higher, 0.2 mg/dl

or higher,

0.3 mg/dl or higher

Interleukin-1
High compared

at least 1%
at least 1 to

receptor
to average level

lower
about 95%

antagonist (IL-1
of normal

lower

ra)
subject

sPLA2(Secretory
High compared

at least 1%
at least 1 to

phospholipase
to average level

lower
about 95%

A2)
of normal

lower

group II A:
subject

type2A, type II A

sPLA2 activity
Low compared

No worsening

to average level

of normal

subjects

Interleukin 2(IL-
High compared

at least 1%
at least 1 to

2)
to average level

lower
about 95%

of normal

lower

subject

ApolipoproteinA-
High compared

at least 1%
at least 1 to

IV
to average level

lower
about 95%

of normal

lower

subject

ApolipoproteinC-
High compared

at least 1%
at least 1 to

II
to average level

lower
about 95%

of normal

lower

subject

CCL2:
High compared

at least 1%
at least 1 to

Chemokine(C-C
to average level

lower
about 95%

motif) ligand 2
of normal

lower

subject

Thrombospondin 1:
High compared

at least 1%
at least 1 to

TSP1
to average level

lower
about 95%

of normal

lower

subject

IL-3 receptor
High compared

at least 1%
at least 1 to

(Interleukin-3
to average level

lower
about 95%

receptor) alpha
of normal

lower

chain
subject

Lymphocyte
High compared

at least 1%
at least 1 to

antigen 6
to average level

lower
about 95%

comlex, locus D
of normal

lower

subject

MMP12:
High compared

at least 1%
at least 1 to

Matrix
to average level

lower
about 95%

metallopeptidase
of normal

lower

12
subject

MMP13:
High compared

at least 1%
at least 1 to

Matrix
to average level

lower
about 95%

metallopeptidase
of normal

lower

13
subject

Trehalase
High compared

at least 1%
at least 1 to

(brush-border
to average level

lower
about 95%

membrane
of normal

lower

glycoprotein)
subject

TIMP1:
High compared

at least 1%
at least 1 to

Tissue inhibitor
to average level

lower
about 95%

of
of normal

lower

metalloproteinase 1
subject

COL1a1:
High compared

at least 1%
at least 1 to

Procollagen type
to average level

lower
about 95%

I, alpha 1
of normal

lower

subject

Complement
High compared

at least 1%
at least 1 to

factor D
to average level

lower
about 95%

(adipsin)
of normal

lower

subject

TNFR (tumor
High compared

at least 1%
at least 1 to

necrosis factor
to average level

lower
about 95%

receptor)
of normal

lower

superfamily,
subject

member 19

(TAJ)

TNFAIP (tumor
High compared

at least 1%
at least 1 to

necrosis factor
to average level

lower
about 95%

alpha induced
of normal

lower

protein) 6
subject

VLDLR (Very low
High compared

at least 1%
at least 1 to

density
to average level

lower
about 95%

lipoprotein
of normal

lower

receptor)
subject

Lipoprotein
High compared

at least 1%
at least 1 to

lipase
to average level

lower
about 95%

of normal

lower

subject

Ear (Eosinophil
High compared

at least 1%
at least 1 to

associated
to average level

lower
about 95%

ribonuclease) A
of normal

lower

family,
subject

members 1,2,3,

and 12

INSL5: Insulin
Low compared

At least 1%

like 5
to average level

increase

of normal

subjects

TGF β2:
Low compared

At least 1%

Transforming
to average level

increase

growth factor
of normal

beta 2
subjects

HAMP:
Low compared

At least 1%

Hepcidin
to average level

increase

antimicrobial
of normal

peptide 1
subjects

Lipase member
Low compared

At least 1%

H:
to average level

increase

LIPH
of normal

subjects

CYP7B1:
Low compared

At least 1%

Cytochrome
to average level

increase

P450 family 7
of normal

subfamily b
subjects

polypeptide 1

TABLE 2

Pre-treatment baseline
After dosing (effect) values

Item (Typical

Observable

Observable

Normal
Typical
Ranges or

Ranges or

Values, Units)
Range(s)
Values
Typical Range(s)
Values

11-HETE
High compared

at least 1%
at least 1 to

(11-hydroxy-
to average level

lower
about 95%

5,8,12,14-
of normal

lower

eicosatetraenoic
subject

acid)

Total HEPEs
Low compared

At least 1%

(hydroxyeicosapentaenoic
to average level

increase

acids)/
of normal

total HETEs
subjects

(Hydroxyeicosatetraenoic

Acids)

ratio

Glycocholate
High compared
Twice or more
at least 1% lower

to average level
than twice as high

of normal
as normal subject

subject

Taurocholate
High compared
Twice or more
at least 1% lower

to average level
than twice as high

of normal
as normal subject

subject

Glycocholate/Glycine
High compared
Twice or more
at least 1% lower

ratio
to average level
than twice as high

of normal
as normal subject

subject

Taurocholate/Taurine
High compared
Twice or more
at least 1% lower

ratio
to average level
than twice as high

of normal
as normal subject

subject

Total fatty acids
Low compared

At least 1%

of 20 to 24
to average level

increase

carbon atoms
of normal

(C20-24)/total
subjects

fatty acids of 16

carbon atoms

(C16) ratio (ex.

μg/ml/μg/ml,

wt %/wt %)

Total omega-3
Low compared

At least 1%

polyunsaturated
to average level

increase

fatty acids of
of normal

20 to 24 carbon
subjects

atoms(C20-24)/

total fatty

acids of 16

carbon atoms

(C16) ratio (ex.

μg/ml/μg/ml,

wt %/wt %)

Total fatty acids
Low compared

At least 1%

of 20 to 24
to average level

increase

carbon
of normal

atoms(C20-24)/
subjects

total fatty

acids of 18

carbon atoms

(C18) ratio (ex.

μg/ml/μg/ml,

wt %/wt %)

Total omega-3
Low compared

At least 1%

polyunsaturated
to average level

increase

fatty acids of
of normal

20 to 24 carbon
subjects

atoms(C20-24)/

total weight of

fatty acids of 18

carbon atoms

(C18) ratio (ex.

μg/ml/μg/ml,

wt %/wt %)

IL-
No change

At least 1%

10(Interleukin-
or Low

increase

10)
compared to

average level of

normal subjects

Small dense LDL
No change
at least 20 mg/dl
at least 1% lower

or High
25, 30, 40, 50, at

compared to
least 60 mg/dl

average level of

normal subjects

RLP-TG
No change
at least 10 mg/dl,
at least 1% lower

(Remnant-like
or High
20, 30, 40, 50, 70,

lipoprotein
compared to
80, 100, 120, at

particles-
average level of
least 150 mg/dl

triglyceride)
normal subjects

RLP-C
No change
At least 4.5 mg/dl,
at least 1% lower,

(Remnant-like
or High
5, 5.2, 5.5, 6, 8,
or no change

lipoprotein
compared to
10, 12, at least 15 mg/dl

particles-
average level of

cholesterol)
normal subjects

Whole Blood

High compared to
at least 1% lower
at least 1% lower

viscosity

average level of

(cP/mPa ·

normal subject

s)

Plasma viscosity

High compared to
No worsening

(cP/mPa ·

average level of

s)

normal subject

IL-10
Low compared

At least 1%

(Interleukin-10)/
to average level

increase

TNFα ratio
of normal

subject

IL-10
Low compared

At least 1%

(Interleukin-10)/
to average level

increase

sCD40 ratio
of normal

subject

Serum
Low compared

At least 1%

adiponectin/
to average level

increase

TNFα ratio
of normal

subject

Serum
Low compared

At least 1%

adiponectin/
to average level

increase

sCD40 ratio
of normal

subject

TABLE 3

11-HETE
Prostaglandins Other Lipid Mediat. 2011 Apr; 94(3-4): 81-7.

HETE, HEPE
Analysis of omega-3 and omega-6 fatty acid-derived lipid

metabolite formation in human and mouse blood samples.

Glycocholate
Metabolism. 2011 Mar; 60(3): 404-13.

Taurocholate
Plasma metabolomic profile in nonalcoholic fatty liver disease.

IL-10
Obes Surg. 2010 Jul; 20(7): 906-12.

Pro- and anti-inflammatory cytokines in steatosis and

steatohepatitis.

Small dense LDL
Diabetol Metab Syndr. 2012 Jul 18; 4(1): 34.

Fatty liver in men is associated with high serum levels of small,

dense low-density lipoprotein cholesterol.

RLP-TG
Clinica Chimica Acta 413 (2012) 1077-1086

RLP-C
The characteristics of remnant lipoproteins in the fasting and

postprandial plasma.

Connective Tissue Growth
Regul Pept. 2012 Sep 4; 179(1-3): 10-14.

Factor (CTGF)
Connective tissue growth factor level is increased in patients

with liver cirrhosis but is not associated with complications or

extent of liver injury.

Serum Soluble CD40 (sCD40)
Apoptosis 2004; 9: 205-210

Role of circulating soluble CD40 as an apoptotic marker in liver

disease.

Complement factor D
Int Immunopharmacol. 2009 Nov; 9(12): 1460-3.

Serum adipsin levels in patients with seasonal allergic rhinitis:

preliminary data.

CK18 fragment
Aliment Pharmacol Ther. 2010 Dec; 32(11-12): 1315-22.

A new composite model including metabolic syndrome, alanine

aminotransferase and cytokeratin-18 for the diagnosis of non-

alcoholic steatohepatitis in morbidly obese patients.

Serum High mobility group
PLoS One. 2012; 7(4): e34318.

box 1 protein (HMGB1)
Diagnostic significance of serum HMGB1 in colorectal

carcinomas.

fatty acid amount and fatty
Clinical Nutrition (2002) 21 (3) 219-223

acid composition ratio in
Plasma total and free fatty acids composition in human non-

blood
alcoholic steatohepatitis.

Ferritin, Thioredoxin
J Hepatol. 2003 Jan; 38(1): 32-8.

Serum thioredoxin levels as a predictor of steatohepatitis in

patients with nonalcoholic fatty liver disease.

sTNF-R1, sTNF-R2
Diabetes Care. 2010 Oct; 33(10): 2244-9. Epub 2010 Jul 27.

Association between systemic inflammation and incident

diabetes in HIV-infected patients after initiation of antiretroviral

therapy.

Hs-CRP
J Hepatol. 2011 Sep; 55(3): 660-5.

C-reactive protein levels in relation to various features of non-

alcoholic fatty liver disease among obese patients.

soluble Fas antigen (CD95,
J Transl Med. 2009 Jul 29; 7: 67.

sFas)
Short term effects of milrinone on biomarkers of necrosis,

apoptosis, and inflammation in patients with severe heart

failure.

Whole Blood viscosity
British Journal of Haematology, 1997 96, 168-173

Plasma viscosity
Blood viscosity and risk of cardiovascular events: the Edinburgh

Artery Study

Items in Table1(1-8, 1-9, 1-10)
WO2011/046204

Example
Treatment of NASH

To evidence the usefulness of the present invention for the treatment of NASH, patients are evaluated for inclusion in the treatment regimen, treated for NASH, and evaluated for effectiveness of the treatment as follows:

Patients are histologically diagnosed with NASH within six months of the initiation of treatment and are willing to submit to a further liver biopsy at the end of the treatment regimen to evaluate effectiveness of the treatment.

1. Inclusion Criteria:

Patients are definitively diagnosed with NASH (via liver biopsy) and exhibit a NAS score of greater than or equal to 4 by a pathologist.

Patients can be of either gender but are greater than 18 years of age.

Patients with diabetes, impaired glucose tolerance or metabolic syndrome that have been on stable dosage of anti-diabetic agents for at least six months prior to the liver biopsy are suitable for treatment.

2. Exclusion Criteria:

Patients may be excluded for treatment based upon an inability or unwillingness to have a liver biopsy for confirming the diagnosis of NASH, having a diagnosis of cirrhosis by pathologist, exhibiting previous bariatric surgery or biliary diversion (i.e. gastric bypass), esophageal banding or gastric banding; serum ALT values of greater than 330 UL, drug use associated with steatohepatitis within 6 months prior to initiation of treatment, such as with corticosteroids, high dose estrogens, methodtrexate, amiodarone, anti-HIV drugs, tamoxifen, or diltiazem; alcohol consumption of greater than 30 g/day, concurrently or for more than three consecutive months within five years prior treatment; a blood alcohol level greater than 0.02% at the time of baseline evaluation; evidence of active substance abuse; including prescription or recreational drugs, the presence of other liver diseases such as acute or chronic hepatitis C, acute or chronic active hepatitis B, Wilson's, autoimmune, alpha1-antitrypsin and hemochromatosis or HIV infection; renal insufficiency; symptomatic coronary; peripheral or neurovascular disease; symptomatic heart failure or advanced respiratory disease requiring oxygen therapy; a history of cerebral or retinal hemorrhage or other bleeding diathesis.

3. Key Criteria for Measuring Baseline and Post Treatment Values:

Patients to be treated are evaluated for one or more of the following criteria.

a) Primary Long-Term Efficacy Outcome Measure

Histology at treatment month 12.5 to evaluate the NAS score, as a comparison to the baseline score measured pre-treatment. (NAS)

b) Primary Short-Term Efficacy Outcome Measure

Change from baseline in ALT levels at Month 3 and Month 6 of treatment.

c) Secondary Efficacy Outcome Measures

Overall NAS score

Feature scores including fibrosis, ballooning degeneration, inflammation and steatosis

Liver function tests (AST, alkaline phosphataise, bilirubin, GGT, Albumin)

Cholesterol (including HDL and LDL)

Triglycerides

Fatty acid assay

Ferritin

Thioredoxin

Pro-inflammatory cytokines (TNF-alpha, sTNF-R1, sTNF-R2, Hs-CRP, CTGF, sCD40)

Insulin sensitivity (HOMA-IR)

HbAlc

Glucose

Leptin, Serum adiponectin and complement factor D

CK18 fragment and Serum HMGB1

soluble Fas antigen

Hyaluronic acid

Type IV collagen (7S domain)

Procollagen III peptide

d) Safety Outcome Measures

Adverse Events

Hematology/biochemistry/urinalysis

ECG (including QT/QTc measurement)

e) Pharmacokinetic Outcome Measures

EPA, DPA and DHA

Day 1

On Day 1, samples for plasma concentration are obtained at predose and 0.5, 1, 2, 4, 5 and 6 hours after Dose #1 and Dose #3; after Dose #2, samples are obtained at 2, 4, 5 and 6 hours post-dose. After Dose #3, samples are also obtained at 8 and 12 hours post-dose (20 and 24 hours after Dose #1 [prior to the morning dose on Day 2]) C_max(Dose #1 and Dose #2s) and C_max, T_max, T_1/2, AUG_0-4after third Dose are derived from plasma concentrations

Days 29, 85, 169 and 365 (Visits 3, 5, 7 and 9)

A single sample is obtained prior to the morning dose (trough) on Visits 3, 5, 7 and 9.

Css is determined from plasma concentrations

4. Concomitant and Medications:

Particular medications can be prohibited or permitted during treatment according to the invention for NASH.

The following medications can be prohibited during treatment:

Omega-3-acid ethyl esters and omega-3-PUFA containing supplements >200 mg per day

Vitamin E>60 IU per day

Thiazolidinediones (e.g. pioglitazone, rosiglitazone)

The following medications may be used during the treatment according to the specified restrictions:

Subjects may continue prescription or over-the-counter medications or herbal remedies such as HMG-CoA reductase inhibitors (stains), fibrates, probucol, ezetimibe, ursodiol (UDCA), taurine, betaine, N-acetylcysteine, s-adenosylmethionine (SAM-e), milk thistle, anti-TNF therapies, or probiotics

Subjects may continue the following anti-diabetic medications: biguanides (metformin), insulin, sulfonylureas, alpha-glucosidase inhibitors (acarbose), dipeptidyl-peptidase 4 inhibitors (sitagliptin, saxagliptin), and phenylalanine derivatives (nateglinide, repaglinide)

Subjects may continue receiving anti-platelet therapy and anti-thrombotic agents (e.g. warfarin, Aspirin (ASA), and clopidogrel) after study commencement should be monitored closely during the study for bleeding problems.

5. Treatment

Patients are treated with EPA-E comprised of two daily treatments, but the total daily dose of EPA-E being 1800 mg or 2700 mg per day, divided into dosage amounts of 600 mg TID or 900 mg TID, respectively.

Treatment with EPA-E is continued for 12 months.

Patients are periodically evaluated for the selected criteria, such as at month 1, month 3, month 6 and month 12 of treatment.

After 12 months of treatment, patients are evaluated for the criteria noted above, including liver biopsy, NAS score, steatosis, lobular inflammation, ballooning and fibrosis stage, and one or more of the other criteria listed above in Tables 1 and 2.

The invention being thus described, it will be apparent to one of ordinary skill in the art that various modifications of the materials and methods for practicing the invention can be made. Such modifications are to be considered within the scope of the invention as defined by the following claims.

Each of the references from the patent and periodical literature cited herein is hereby expressly incorporated in its entirety by such citation.

	Number	Date	Country
Parent	14435121	Apr 2015	US
Child	15269134		US

Compositions and Methods for Treating Non-Alcoholic Steatohepatitis

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