HK1246191B - Pharmaceutical compositions for combination therapy - Google Patents

Description

Pharmaceutical compositions for combination therapy

Background Art

The concentration of circulating lipid compounds (such as cholesterol and triglycerides) in the blood increases with many diseases. These diseases include type ii diabetes, primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), various chronic hepatitis states (type B and C hepatitis), NASH (non-alcoholic steatohepatitis) and arterial disease (including coronary artery disease, cerebrovascular arterial disease, peripheral vascular disease, aortic aneurysm and carotid atherosclerosis disease). Various lipid-lowering technologies have been used in the past to treat and prevent vascular events (such as heart failure, embolism, heart attack and stroke) associated with hyperlipidemia. Such therapeutic methods have included dietary changes and control of the high triglyceride and cholesterol levels circulating in the blood. The latter is usually pharmacologically and recently treated with various "statins". Included in the therapeutic agent for the disease that is used to treat lipid levels is various fibric acid derivatives. Some older fibric acid derivatives including clofibrate have occupied a past position in the illness relevant to lipid elevation in treatment, but recent new fibrates (including fenofibrate, gemfibrozil, ciprofibrate) and even closer fibrates (including piperidine, 4-hydroxypiperidine, piperidine-3-ene and piperazine) have joined the ranks of anti-lipid therapy.These newer molecules have promising characteristics and reduce both cholesterol and triglyceride.Yet, in some cases, independent fibric acid derivatives are not enough to control the serious hyperlipidemia level existing in many patients.The side effect profile of fibric acid derivatives also can be improved from the dosage reduction such as in the presence of combination therapy.

Therefore, there is a need for improved therapies for treating conditions involving elevated concentrations of circulating lipid compounds, such as cholesterol and triglycerides, in the blood.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1A is a representative photomicrograph of Sirius-red stained liver sections from sham BDL mice.

FIG. 1B is a representative photomicrograph of Sirius red-stained liver sections from BDL vehicle-treated mice.

FIG1C is a representative photomicrograph of Sirius red-stained liver sections from BDL-OCA-treated mice.

FIG. 1D is a representative photomicrograph of Sirius red-stained liver sections from BDL-atorvastatin-treated mice.

FIG. 1E is a representative photomicrograph of Sirius red-stained liver sections from BDL-OCA-atorvastatin-treated mice.

FIG2 is a bar graph showing Sirius Red-positive area (%) in BDL mice treated with OCA and atorvastatin alone and in combination.

FIG3A is a bar graph showing the number of inflammatory cell foci from APOE*3Leiden.CETP mice treated with OCA, low-dose fenofibrate, alone and in combination.

FIG3B is a bar graph showing the number of inflammatory cell foci from APOE*3Leiden.CETP mice treated with OCA, high-dose fenofibrate, alone and in combination.

4 is a bar graph showing the effects of OCA and atorvastatin, alone and in combination, on the stage of fibrosis in leptin-ob/ob mice.

FIG5A is a bar graph showing plasma triglyceride levels in leptin-ob/ob mice treated with OCA and atorvastatin alone and in combination.

Figure 5B is a bar graph showing the change from baseline in plasma triglyceride levels in leptin-ob/ob mice treated with OCA and atorvastatin alone and in combination.

FIG6A is a bar graph showing enrichment analysis of canonical pathways in HFC+OCA versus HFC-maintained APOE*3Leiden.CETP mice.

FIG6B is a bar graph showing enrichment analysis of canonical pathways in HFC+OCA+low-dose fenofibrate versus HFC-maintained APOE*3Leiden.CETP mice.

FIG7A is a Venn diagram showing the number of novel differentially expressed genes modulated by the combination of OCA + low-dose fenofibrate versus monotherapy in APOE*3Leiden.CETP mice.

FIG7B is a bar graph showing pathway enrichment of genes regulated by the combination of OCA + low-dose fenofibrate relative to monotherapy in APOE*3Leiden.CETP mice.

FIG8 is a graph showing the effects of OCA and statins in combination on LDL cholesterol in humans.

Summary of the Invention

The present application relates to a pharmaceutical composition comprising (i) a first compound, (ii) at least one PPAR-α agonist, PPAR-δ agonist and/or PPAR-α and δ dual agonist, and (iii) optionally one or more pharmaceutically acceptable carriers, wherein the first compound is a FXR agonist.

The present invention also relates to a pharmaceutical composition comprising (i) a first compound, (ii) at least one fibrate, and optionally (iii) one or more pharmaceutically acceptable carriers, wherein the first compound is a FXR agonist.

The present invention also relates to a pharmaceutical composition comprising (i) a first compound, (ii) at least one lipid-lowering agent, and optionally (iii) one or more pharmaceutically acceptable carriers, wherein the first compound is a FXR agonist.

The present invention also relates to a pharmaceutical composition comprising (i) a first compound, (ii) at least one statin, and optionally (iii) one or more pharmaceutically acceptable carriers, wherein the first compound is a FXR agonist.

The present invention also relates to a pharmaceutical composition comprising (i) a first compound, (ii) at least one PPAR-α agonist, PPAR-δ agonist and/or PPAR-α and δ dual agonist, (iii) at least one lipid-lowering agent, and optionally (iv) one or more pharmaceutically acceptable carriers, wherein the first compound is a FXR agonist.

The present invention also relates to a pharmaceutical composition comprising (i) a first compound, (ii) at least one fibrate, (iii) at least one lipid-lowering agent, and optionally (iv) one or more pharmaceutically acceptable carriers, wherein the first compound is a FXR agonist.

The present invention also relates to a pharmaceutical composition comprising (i) a first compound, (ii) at least one PPAR-α agonist, PPAR-δ agonist and/or PPAR-α and δ dual agonist, (iii) at least one statin, and optionally (iv) one or more pharmaceutically acceptable carriers, wherein the first compound is a FXR agonist.

The present invention also relates to a pharmaceutical composition comprising (i) a first compound, (ii) at least one fibrate, (iii) at least one statin, and optionally (iv) one or more pharmaceutically acceptable carriers, wherein the first compound is a FXR agonist.

The present invention also relates to the therapeutic use of the pharmaceutical composition of the present invention.

In one embodiment, the first compound is a compound of Formula A:

or a pharmaceutically acceptable salt or amino acid conjugate thereof, wherein R ₁ , R ₂ , R ₄ , R ₇ and X are as defined herein.

The present invention also relates to methods for treating or preventing FXR-mediated diseases or conditions or diseases or conditions involving elevated concentrations of circulating lipid compounds in the blood, for lowering liver enzyme levels, or inhibiting or reversing fibrosis, which methods comprise administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of the present invention.

The present invention also relates to the use of the pharmaceutical compositions of the present invention for treating or preventing FXR-mediated diseases or conditions or diseases or conditions involving elevated concentrations of circulating lipid compounds in the blood, for reducing liver enzyme levels, or for inhibiting or reversing fibrosis.

The present invention also relates to the use of the pharmaceutical composition of the present invention in the manufacture of a medicament for treating or preventing FXR-mediated diseases or conditions or diseases or conditions involving elevated concentrations of circulating lipid compounds in the blood, for lowering liver enzyme levels, or for inhibiting or reversing fibrosis.

The compositions and methods of the present invention address an unmet need in the treatment or prevention of diseases or disorders in which elevated concentrations of circulating lipid compounds, such as cholesterol and triglycerides, in the blood are involved.

DETAILED DESCRIPTION

The present application relates to a pharmaceutical composition comprising a first compound, at least one PPAR-α agonist, PPAR-δ agonist, and/or PPAR-α and δ or PPAR-α and γ dual agonist, and optionally one or more pharmaceutically acceptable carriers, wherein the first compound is a FXR agonist.

In one example, the pharmaceutical composition comprises at least one PPAR-α agonist. In one example, the pharmaceutical composition comprises at least one PPAR-δ agonist. In one example, the pharmaceutical composition comprises at least one PPAR-α and δ dual agonist. In one example, the pharmaceutical composition comprises at least one PPAR-α and γ dual agonist. In one example, the pharmaceutical composition comprises at least one PPAR-α agonist and at least one PPAR-δ agonist. In one example, the pharmaceutical composition comprises at least one PPAR-α agonist and at least one PPAR-α and δ dual agonist. In one example, the pharmaceutical composition comprises at least one PPAR-δ agonist and at least one PPAR-α and δ or PPAR-α and γ dual agonist. In one example, the pharmaceutical composition comprises at least one PPAR-α agonist, at least one PPAR-δ agonist, and at least one PPAR-α and δ dual agonist. In one example, the PPAR-α agonist is a fibrate, such as a fibrate described herein. In one example, the PPAR-δ agonist is {4-[({4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl}methyl)sulfanyl]-2-methylphenoxy}acetic acid (also known as GW501516, GW1516, and "Endurabol"), {2-methyl-4-[5-methyl-2-(4-trifluoromethyl-phenyl)-2H-[1,2,3]triazol-4-ylmethylsulfanyl]-phenoxy}-acetic acid, or [4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphenoxy]-acetic acid or a pharmaceutically acceptable salt thereof. In one example, the PPAR-α and δ dual agonist is 2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1(E)-propenyl]phenoxy]-2-methylpropanoic acid (also known as GFT505). In one embodiment, the PPAR-α and γ dual agonist is aleglitazar ((2S)-2-methoxy-3-[4-[2-(5-methyl-2-phenyl-4-oxazolyl)ethoxy]-7-benzophenylthio]propionic acid), moglitazar (N-[(4-methoxyphenoxy)carbonyl]-N-{4-[2-(5-methyl-2-phenyl-1,3-oxazol-4-yl)ethoxy]benzyl}glycine), tesaglitazar ((2S)-2-ethoxy-3-[4-[2-(4-methylsulfonyloxyphenyl)ethoxy]phenyl]propionic acid), or saroglitazar ((2S)-2-ethoxy-3-[4-(2-{2-methyl-5-[4-(methylsulfanyl)phenyl]-1H-pyrrol-1-yl}ethoxy)phenyl]propionic acid), or a pharmaceutically acceptable salt thereof. In one example, the PPAR-α and δ dual agonist is 2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1(E)-propenyl]phenoxy]-2-methylpropanoic acid or a pharmaceutically acceptable salt thereof.

The present application also relates to a pharmaceutical composition comprising a first compound, at least one fibrate, and optionally one or more pharmaceutically acceptable carriers, wherein the first compound is a FXR agonist. The FXR agonist can be any FXR agonist. The fibrate can be any fibrate. In one example, the fibrate is selected from any of the fibrates described herein.

The present application also relates to a pharmaceutical composition comprising a first compound, at least one lipid-lowering agent, and optionally one or more pharmaceutically acceptable carriers, wherein the first compound is a FXR agonist. The FXR agonist can be any FXR agonist. The lipid-lowering agent can be any lipid-lowering agent. In one example, the lipid-lowering agent is selected from any of the lipid-lowering agents described herein.

The present application also relates to a pharmaceutical composition comprising a first compound, at least one statin, and optionally one or more pharmaceutically acceptable carriers, wherein the first compound is a FXR agonist. The FXR agonist can be any FXR agonist. The statin can be any statin. In one example, the statin is selected from any statin described herein.

The present application also relates to a pharmaceutical composition comprising a first compound, at least one PPAR-α agonist, PPAR-δ agonist, and/or a dual agonist of PPAR-α and δ or PPAR-α and γ, at least one lipid-lowering agent, and optionally one or more pharmaceutically acceptable carriers, wherein the first compound is a FXR agonist. The present application also relates to a pharmaceutical composition comprising a first compound, at least one PPAR-α agonist, PPAR-δ agonist, and/or a dual agonist of PPAR-α and δ or PPAR-α and γ, at least one statin, and optionally one or more pharmaceutically acceptable carriers, wherein the first compound is a FXR agonist. In one example, the PPAR-α agonist is a fibrate, such as a fibrate described herein. In one example, the PPAR-δ agonist is {4-[({4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl}methyl)sulfanyl]-2-methylphenoxy}acetic acid, {2-methyl-4-[5-methyl-2-(4-trifluoromethyl-phenyl)-2H-[1,2,3]triazol-4-ylmethylsulfanyl]-phenoxy}-acetic acid, or [4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphenoxy]-acetic acid, or a pharmaceutically acceptable salt thereof. In one example, the PPAR-α and δ dual agonist is 2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1(E)-propenyl]phenoxy]-2-methylpropanoic acid. In one example, the PPAR-α and γ dual agonist is aleglitazar, moglitazole, tesaglitazar, or sarcoglitazar, or a pharmaceutically acceptable salt thereof. In one example, the PPAR-α and δ dual agonist is 2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1(E)-propenyl]phenoxy]-2-methylpropanoic acid, or a pharmaceutically acceptable salt thereof. In one example, the lipid-lowering agent is selected from any lipid-lowering agent described herein. In one example, the statin is selected from any statin described herein.

In one example, the first compound of the pharmaceutical composition is a compound having Formula A:

or a pharmaceutically acceptable salt or amino acid conjugate thereof, wherein:

_R1 is hydrogen or unsubstituted _C1 - _C6 alkyl;

_R2 is hydrogen or α-hydroxy;

X is C(O)OH, C(O)NH(CH ₂ ) _m SO ₃ H, C(O)NH(CH ₂ ) _n CO ₂ H or OSO ₃ H;

_R4 is hydroxy or hydrogen;

_R7 is hydroxy or hydrogen;

m is 1, 2, or 3; and

n is 1, 2, or 3.

In another embodiment, the first compound of the pharmaceutical composition is selected from Formula I and IA:

or a pharmaceutically acceptable salt or amino acid conjugate thereof, wherein

R _1A is hydrogen or unsubstituted C ₁ -C ₆ alkyl;

_R2 is hydrogen or α-hydroxy;

_R4 is hydroxy or hydrogen; and

_R7 is hydroxy or hydrogen.

In one aspect, the first compound is a pharmaceutically acceptable salt. In one embodiment, the first compound is a sodium salt of Formula I or IA. In another embodiment, the first compound is an ammonium salt of a compound of Formula I or IA. In another embodiment, the first compound is a triethylammonium salt of a compound of Formula I or IA.

In yet another example, the first compound of the pharmaceutical composition is selected from Formula II and IIA:

or a pharmaceutically acceptable salt or amino acid conjugate thereof, wherein:

R _1A is hydrogen or unsubstituted C ₁ -C ₆ alkyl;

_R2 is hydrogen or α-hydroxy;

R ₃ is hydroxy, NH(CH ₂ ) _m SO ₃ H or NH(CH ₂ ) _n CO ₂ H;

_R4 is hydroxy or hydrogen;

_R7 is hydroxy or hydrogen;

m is 1, 2, or 3; and

n is 1, 2, or 3.

In one example, the composition includes a first compound having Formula A, I, IA, II, or IIA, wherein R ₂ is hydrogen.

In another example, the composition includes a first compound of Formula A, wherein _R1 is an unsubstituted _C1 - _C6 alkyl. In one aspect, the composition includes a first compound of Formula A, wherein _R1 is an unsubstituted C1- _C3 alkyl. In one aspect, the composition includes a first compound of Formula A, wherein _R1 is selected from methyl, _ethyl , and propyl. In one aspect, the composition includes a first compound of Formula A, wherein _R1 is ethyl.

In another example, the composition includes a first compound of Formula I, IA, II, or IIA, wherein R _1A is unsubstituted C ₁ -C ₆ alkyl. In one aspect, the composition includes a first compound of Formula I, IA, II, or IIA, wherein R _1A is unsubstituted C ₁ -C ₃ alkyl. In one aspect, the composition includes a first compound of Formula I, IA, II, or IIA, wherein R _1A is selected from methyl, ethyl, and propyl. In one aspect, the composition includes a first compound of Formula I, IA, II, or IIA, wherein R _1A is ethyl.

In another example, the composition includes a first compound of formula A, wherein X is selected from C(O)OH, C(O)NH(CH ₂ ) _m SO ₃ H, and C(O)NH(CH ₂ ) _n CO ₂ H. In one aspect, the composition includes a first compound of formula A, wherein X is selected from C(O)OH, C(O)NH(CH ₂ ) SO ₃ H, C(O)NH(CH ₂ ) CO ₂ H, C(O)NH(CH ₂ ) ₂ SO ₃ H, C(O)NH(CH ₂ ) ₂ CO ₂ H. In one aspect, the composition includes a first compound of formula A, wherein X is C(O)OH. In one aspect, the composition includes a first compound of formula A, wherein X is OSO ₃ H. In one aspect, the composition includes a first compound of formula A, wherein the first compound is a pharmaceutically acceptable salt. The pharmaceutically acceptable salt can be any salt. In one aspect, the composition comprises a first compound of formula A, wherein X is OSO ₃ ⁻ Na ⁺ . In one aspect, the composition comprises a first compound of formula A, wherein X is OSO ₃ ⁻ NHEt ₃ ⁺ . In one aspect, the amino acid conjugate is a glycine conjugate. In one aspect, the amino acid conjugate is a taurine conjugate.

In yet another example, the composition includes a first compound having Formula II or IIA, wherein R ₃ is selected from OH, NH(CH ₂ )SO ₃ H, NH(CH ₂ )CO ₂ H, NH(CH ₂ ) _{2SO 3} H, and NH(CH ₂ ) _{2CO 2} H. In one aspect, the composition includes a first compound having Formula II or IIA, wherein R ₃ is OH.

In further examples, the composition includes a first compound of Formula A, I, or II, wherein R ₄ is hydroxy and R ₇ is hydrogen.

In another example, the composition includes a first compound of Formula A, wherein R ₁ is selected from methyl, ethyl, and propyl, R ₄ is OH, R ₇ is H, and R ₂ is H.

In further examples, the composition includes a first compound of Formula I or II, wherein R _1A is selected from methyl, ethyl, and propyl, R ₄ is OH, R ₇ is H, and R ₂ is H.

In further examples, the composition includes a first compound of Formula IA or IIA, wherein R _1A is selected from methyl, ethyl, and propyl, and R ₂ is H.

In another example, the composition includes a first compound selected from the group consisting of

or a pharmaceutically acceptable salt or amino acid conjugate thereof.

In yet other examples, the composition includes a first compound selected from the group consisting of

Compounds of Formula I, IA, II, and IIA are subsets of compounds of Formula A. The features described herein for compounds of Formula A also apply to compounds of Formula I, IA, II, and IIA. This application also describes pharmaceutical compositions, packages or kits, and therapeutic uses of the combinations.

One of the problems to be solved by the present invention is to identify combination therapies for treating or preventing conditions associated with elevated concentrations of circulating lipid compounds (such as cholesterol and triglycerides) in the blood (e.g., cholestatic liver conditions such as PBC), and for reducing circulating lipid compounds (e.g., cholesterol, LDL, and triglycerides) in the blood, and for reducing bilirubin and/or liver enzymes (e.g., alkaline phosphatase (ALP, AP, or Alk Phos), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), lactate dehydrogenase (LDH), and 5' nucleotidase). Although drugs for conditions associated with elevated lipid and/or liver enzyme levels are available, these drugs are often unsuitable for many patients for various reasons. For example, certain drugs are ineffective for patients who have developed resistance to, for example, ursodeoxycholic acid. As another example, many statins have adverse effects such as muscle problems, cognitive loss, neuropathy, pancreatic and liver dysfunction, and sexual dysfunction. When given alone, some drugs may be insufficient for treatment. In some cases, a kind of lipid-lowering agent is not enough to control the serious hyperlipidemia level existing in many patients.Some medicines may need high doses of giving or more frequent giving due to being widely metabolized into inactive or lower metabolites of effectiveness.Combination therapy described herein can solve the above problems and can have one or more following advantages: for example, synergy, reduces the number of times of daily dosage when there is no drug loss effect, reduces lipid (cholesterol and triglyceride) in the patient of the treatment in the lipid level resistance PBC of rising, improved effectiveness, selectivity, tissue permeability, half-life and/or metabolic stability.

In the compositions, packages or kits, methods, and uses of the present invention, the first compound can be a free acid or the first compound can be a pharmaceutically acceptable salt or amino acid conjugate (e.g., a glycine or taurine conjugate). In one aspect, the first compound is any FXR agonist. In one aspect, the first compound is a compound of formula A. In one aspect, the first compound is a compound of formula I or IA. In one aspect, the first compound is a compound of formula IA. In one aspect, the first compound is a compound of formula II or IIA. In one aspect, the first compound is a compound of formula IIA. In one aspect, the first compound is obeticholic acid (Compound 1). In one aspect, the first compound is Compound 2. In one aspect, the first compound is a pharmaceutically acceptable salt of Compound 3. In one aspect, the first compound is a pharmaceutically acceptable salt of Compound 4.

In the compositions, packaging or kits, methods and uses of the present invention, the fibrate can be any fibrate. In one aspect, the fibrate is selected from the group consisting of fenofibrate, bezafibrate, benzclofibrate, binifbrate, ciprofibrate, clinofibrate, clofibrate, clofibric acid, etofibrate, gemfibrozil, nicofibrate, pirfibrate, cloniafibrate, bisfibrate, clofibrate, tocofibrate, prafibride and pharmaceutically acceptable salts and esters thereof, and derivatives of 2-phenoxy-2-methylpropionic acid (wherein the phenoxy moiety is substituted with an optionally substituted residue of piperidine, 4-hydroxypiperidine, piperidine-3-ene or piperazine), as disclosed in European Patent Application Publication No. EP0607536. In one aspect, the fibrate is selected from the group consisting of bezafibrate, ciprofibrate, clofibrate, fenofibrate, gemfibrozil, binifibrate, clinofibrate, clofibric acid, nicofibrate, pirfibrate, prafibramide, cloniafibrate, clofibrine, tocofibrate and pharmaceutically acceptable salts and esters thereof, and derivatives of 2-phenoxy-2-methylpropionic acid (wherein the phenoxy moiety is substituted with an optionally substituted residue of piperidine, 4-hydroxypiperidine, piperidin-3-ene or piperazine), as disclosed in European Patent Application Publication No. EP0607536. An example of the latter group of substances is 2-[3-[1-(4-fluorobenzoyl)piperidin-4-yl]phenoxy-2-methyl-propionic acid. For example, the fibrate is bezafibrate, fenofibrate, gemfibrozil, ciprofibrate, clofibrate, clofibric acid or a pharmaceutically acceptable salt or ester thereof. For example, the fibrate is fenofibrate or a pharmaceutically acceptable salt selected from choline, ethanolamine, diethanolamine, piperazine, calcium and tromethamine. For example, the fibrate is clofibrate or a pharmaceutically acceptable salt or ester thereof, such as etofibrate or aluminum clofibrate. For example, the fibrate is bezafibrate. For example, the fibrate is a derivative of 2-phenoxy-2-methylpropionic acid, such as 2-[3-[1-(4-fluorobenzoyl)-piperidin-4-yl]phenoxy-2-methylpropionic acid.

In one embodiment, the first compound is the free acid of a compound of formula A, and the at least one fibrate is selected from bezafibrate, fenofibrate, gemfibrozil, ciprofibrate, clofibrate, and pharmaceutically acceptable salts or esters thereof.

In one embodiment, the first compound is a pharmaceutically acceptable salt of a compound of formula A, and the at least one fibrate is selected from bezafibrate, fenofibrate, gemfibrozil, ciprofibrate, clofibrate, and pharmaceutically acceptable salts or esters thereof.

In one embodiment, the first compound is a glycine conjugate of a compound of formula A, and the at least one fibrate is selected from bezafibrate, fenofibrate, gemfibrozil, ciprofibrate, clofibrate, and pharmaceutically acceptable salts or esters thereof.

In one embodiment, the first compound is a taurine conjugate of a compound of formula A, and the at least one fibrate is selected from bezafibrate, fenofibrate, gemfibrozil, ciprofibrate, clofibrate, and pharmaceutically acceptable salts or esters thereof.

In one embodiment, the first compound is a compound of formula A or a pharmaceutically acceptable salt or amino acid conjugate, and the at least one fibrate is 2-[3-[1-(4-fluorobenzoyl)-piperidin-4-yl]phenoxy-2-methylpropionic acid.

In one embodiment, the first compound is a compound of formula A, or a pharmaceutically acceptable salt or amino acid conjugate, and the at least one PPAR-delta agonist is {4-[({4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl}methyl)sulfanyl]-2-methylphenoxy}acetic acid, {2-methyl-4-[5-methyl-2-(4-trifluoromethyl-phenyl)-2H-[1,2,3]triazol-4-ylmethylsulfanyl]-phenoxy}-acetic acid, or [4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphenoxy]-acetic acid, or a pharmaceutically acceptable salt thereof.

In one embodiment, the first compound is a compound of formula A or a pharmaceutically acceptable salt or amino acid conjugate, and the at least one PPAR-α and δ dual agonist is 2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1(E)-propenyl]phenoxy]-2-methylpropanoic acid. In one embodiment, the first compound is a compound of formula A or a pharmaceutically acceptable salt or amino acid conjugate, and the at least one PPAR-α and γ dual agonist is aleglitazar, moglitazar, tesaglitazar, or saroglitazar, or a pharmaceutically acceptable salt thereof. In one example, the PPAR-α and δ dual agonist is 2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1(E)-propenyl]phenoxy]-2-methylpropanoic acid, or a pharmaceutically acceptable salt thereof.

In the compositions, packaging or kits, methods and uses of the present invention, the statin can be any statin. In one aspect, the statin is selected from the group consisting of simvastatin, fluvastatin, pravastatin, rivastatin, mevastatin, atorvastatin, cerivastatin, lovastatin, pitavastatin, fluindostatin, velostatin, dalvavastatin, rosuvastatin, dihydrocompactin and compatin.

In one embodiment, the first compound is the free acid of a compound of formula A, and the at least one statin is selected from simvastatin, fluvastatin, pravastatin, rivastatin, mevastatin, atorvastatin, cerivastatin, lovastatin, pitavastatin, fludostatin, vilvastatin, dalvastatin, rosuvastatin, dihydrocompactin, and compatin.

In one embodiment, the first compound is a pharmaceutically acceptable salt of a compound of formula A, and the at least one statin is selected from simvastatin, fluvastatin, pravastatin, rivastatin, mevastatin, atorvastatin, cerivastatin, lovastatin, pitavastatin, fludostatin, vilvastatin, dalvastatin, rosuvastatin, dihydrocompactin and compatin.

In one embodiment, the first compound is a glycine conjugate of a compound of formula A, and the at least one statin is selected from simvastatin, fluvastatin, pravastatin, rivastatin, mevastatin, atorvastatin, cerivastatin, lovastatin, pitavastatin, fludostatin, vilvastatin, dalvastatin, rosuvastatin, dihydrocompactin, and compatin.

In one embodiment, the first compound is a taurine conjugate of a compound of formula A, and the at least one statin is selected from simvastatin, fluvastatin, pravastatin, rivastatin, mevastatin, atorvastatin, cerivastatin, lovastatin, pitavastatin, fludostatin, vilvastatin, dalvastatin, rosuvastatin, dihydrocompactin, and compatin.

The present invention also includes an isotopically labeled first compound or a pharmaceutically acceptable salt or amino acid conjugate thereof, which has a structure identical to that of the first compound of the present invention (e.g., a compound of Formula A, I, IA, II, or IIA), except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature. Examples of isotopes that can be incorporated into the first compound or a pharmaceutically acceptable salt or amino acid conjugate thereof include isotopes of hydrogen, carbon, nitrogen, fluorine, such as ³ H, ¹¹ C, ¹⁴ C, and ¹⁸ F.

A first compound comprising the above-mentioned isotopes and/or other isotopes of other atoms, or a pharmaceutically acceptable salt thereof, or an amino acid conjugate thereof is within the scope of the present invention. An isotopically labeled first compound, or a pharmaceutically acceptable salt thereof, or an amino acid conjugate thereof, for example, a first compound incorporating a radioactive isotope such as ³ H and/or ¹⁴ C, can be used in drug and/or substrate tissue distribution assays. Tritiated (i.e., ³ H) and carbon-14 (i.e., ¹⁴ C) isotopes are used because of their ease of preparation and detectability. In addition, substitution with heavier isotopes such as deuterium (i.e., ² H) can provide certain therapeutic advantages caused by greater metabolic stability, such as increased in vivo half-life and reduced dosage requirements, and therefore can be used in some cases. An isotopically labeled first compound, or a pharmaceutically acceptable salt thereof, or an amino acid conjugate thereof can generally be prepared by performing the procedures disclosed in the schemes and/or examples of the present invention, by replacing a non-isotopically labeled reagent with an easily available isotopically labeled reagent. In one embodiment, obeticholic acid or a pharmaceutically acceptable salt thereof or an amino acid conjugate thereof is not isotopically labeled.

The present invention also provides a method for treating or preventing a disease or condition, which comprises administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a subject in need thereof.

In one embodiment, the disease or condition is a FXR-mediated disease or condition. Examples of FXR-mediated diseases or conditions include, but are not limited to, liver disease (including cholestatic and non-cholestatic liver disease) (such as primary biliary cirrhosis (PBC)), primary sclerosing cholangitis (PSC), biliary atresia, portal hypertension, bile acid diarrhea, chronic liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatitis C infection, alcoholic liver disease, liver damage due to progressive fibrosis, and liver fibrosis. Examples of FXR-mediated diseases also include hyperlipidemia, high LDL-cholesterol, high HDL-cholesterol, high triglycerides, and cardiovascular disease.

NAFLD is a medical condition characterized by fat accumulation in the liver (known as fatty infiltration). NAFLD is one of the most common causes of chronic liver disease and covers a range of conditions related to lipid deposition in hepatocytes. It ranges from steatosis (simple fatty liver) to non-alcoholic steatohepatitis (NASH) to advanced fibrosis and cirrhosis. The disease is usually silent and is often discovered through occasional elevated liver enzyme levels. NAFLD is closely related to obesity and insulin resistance and is currently considered by many to be the liver component of metabolic syndrome.

Non-alcoholic steatohepatitis (NASH) is a condition that causes inflammation and accumulation of fat and fibrous (scar) tissue in the liver. Liver enzyme levels in the blood may be higher than the slight elevations observed in non-alcoholic fatty liver (NAFL). Although similar conditions can occur in alcoholics, NASH occurs in those who rarely drink to no alcohol. NASH affects 2% to 5% of Americans and is most frequently found in people with one or more of the following conditions: obesity, diabetes, hyperlipidemia, insulin resistance, use of certain medications, and exposure to toxins. NASH is an increasingly common cause of chronic liver disease worldwide and is associated with increased liver-related mortality and hepatocellular carcinoma (even in the absence of cirrhosis). NASH progresses to cirrhosis in 15%-20% of affected individuals and is now one of the main indications for liver transplantation in the United States. Currently, there is no approved therapy for NASH.

In one embodiment, the disease or condition is hyperlipidemia. In one embodiment, the disease or condition is cholestatic liver disease. In one embodiment, the disease or condition is PBC. In another embodiment, the disease or condition is cardiovascular disease. In another embodiment, the cardiovascular disease is arteriosclerosis, hypercholesterolemia, or hypertriglyceridemia.

The present invention also provides methods for treating or preventing NAFLD or NASH. In one embodiment, the present invention provides methods for treating or preventing NAFLD or NASH associated with hyperlipidemia. In one embodiment, the present invention provides methods for treating or preventing NASH. In one embodiment, the present invention provides methods for treating or preventing NASH associated with hyperlipidemia.

The present invention also provides a method for inhibiting or reversing fibrosis, comprising administering a therapeutically effective amount of a pharmaceutical composition of the present invention to a subject in need thereof. In one embodiment, the subject does not suffer from a cholestatic disorder. In another embodiment, the subject suffers from a cholestatic disorder.

In one embodiment, the subject does not have a cholestatic disorder associated with a disease or disorder selected from the group consisting of primary liver cancer and bile duct cancer (including hepatocellular carcinoma), colorectal cancer, metastatic cancer, sepsis, chronic total parenteral nutrition, cystic fibrosis, and granulomatous liver disease. In embodiments, the fibrosis to be inhibited or reversed occurs in an organ expressing FXR.

In one embodiment, cholestatic disease is defined as the serum level of the abnormal increase with alkaline phosphatase, gamma-glutamyl transpeptidase (GGT), and/or 5 ' nucleotidase. In another embodiment, cholestatic disease is further defined as presenting at least one clinical symptom. In one embodiment, the symptom is itching (pruritus). In another embodiment, cholestatic disease is selected from the group consisting of primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), drug-induced cholestasis, hereditary cholestasis, biliary atresia, and intrahepatic cholestasis of pregnancy.

In one embodiment, the fibrosis is selected from the group consisting of liver fibrosis, kidney fibrosis, and intestinal fibrosis.

In one embodiment, the subject has liver fibrosis associated with a disease selected from the group consisting of: hepatitis B; hepatitis C; parasitic liver disease; post-transplant bacterial, viral and fungal infections; alcoholic liver disease (ALD); non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); liver disease induced by methotrexate, isoniazid, phenobarbital, methyldopa, hibak, tolbutamide, or amiodarone; autoimmune hepatitis; sarcoidosis; Wilson's disease; hemochromatosis; Gaucher disease; glycogen storage disease types III, IV, VI, IX and X alpha ₁ -antitrypsin deficiency; cerebrohepatorenal syndrome; tyrosinemia; fructosemia; galactosemia; vascular disorders associated with Budd-Chiari syndrome, veno-occlusive disease, or portal vein thrombosis; and congenital hepatic fibrosis.

In another embodiment, the subject has intestinal fibrosis associated with a disease selected from the group consisting of Crohn's disease, ulcerative colitis, post-radiation colitis, and microscopic colitis.

In another embodiment, the patient has renal fibrosis associated with a disease selected from the group consisting of diabetic nephropathy, hypertensive nephrosclerosis, chronic glomerulonephritis, chronic transplant glomerulopathy, chronic interstitial nephritis, and polycystic kidney disease.

The present invention also provides methods for treating or preventing all forms of conditions associated with elevated lipid levels. In one embodiment, the condition is hyperlipidemia, wherein it is associated with a condition selected from the group consisting of: resistant primary biliary cirrhosis; primary biliary cirrhosis with associated elevated liver function tests and hyperlipidemia, primary sclerosing cholangitis, non-alcoholic steatohepatitis; and chronic liver disease associated with hepatitis B, hepatitis C, or alcoholic hepatitis. In another embodiment, the present invention provides methods for treating or preventing hyperlipidemia, wherein the hyperlipidemia is primary hyperlipidemia with or without a genetic component, or hyperlipidemia associated with coronary artery disease, cerebrovascular arterial disease, peripheral vascular disease, aortic aneurysm, or atherosclerosis.

In one aspect, the present invention provides methods for treating or preventing primary sclerosing cholangitis, in conjunction with similar biochemical abnormalities, chronic hepatitis caused by hepatitis B, hepatitis C, or alcoholic hepatitis. In one aspect, the present invention provides methods for treating or preventing other arterial diseases associated with hyperlipidemia. In one aspect, the present invention provides methods for treating or preventing hypertriglyceridemia.

The present invention also provides a method for reducing the lipid levels (i.e. the amount of lipid) in the blood, the method including giving the pharmaceutical composition of the present invention of a therapeutically effective amount to a subject in need thereof. In one embodiment, compared to control subjects (e.g., the subject not given the composition of the present invention), the method of the present invention reduces lipid levels by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In one embodiment, compared to healthy subjects (e.g., without the individuality of disease or illness (such as those described herein)), the subject has the lipid levels of elevation. In one embodiment, the method of the application reduces lipid levels to normal levels (e.g., similar to the lipid levels in the individuality without disease or illness (such as those described herein)).

In one embodiment, the lipid is cholesterol.In one embodiment, compared to control subjects (for example, the experimenter of the compositions of the present invention is not given), the method of the present invention reduces cholesterol levels by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.In one embodiment, compared to healthy subjects (for example, there is no individuality of disease or illness (as described herein)), the experimenter has the cholesterol levels that rise.In one embodiment, the method of the present invention reduces cholesterol levels to below 400mg/L, 350mg/L, 300mg/L, 250mg/L, 240mg/L, 230mg/L, 220mg/L, 210mg/L, 200mg/L, 190mg/L, 180mg/L, 170mg/L, 160mg/L or 150mg/L. In one embodiment, the methods of the invention reduce cholesterol levels to below 200 mg/L, 190 mg/L, 180 mg/L, 170 mg/L, 160 mg/L, or 150 mg/L.

In one embodiment, the cholesterol is LDL.In one embodiment, compared to control subjects (for example, the subject of the compositions of the present invention is not given), the method of the present invention reduces LDL levels by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.In one embodiment, compared to healthy subjects (for example, the individuality without disease or illness (such as those described herein)), the subject has the LDL levels of elevation.In one embodiment, the method of the present invention reduces LDL levels to below 300mg/L, 200mg/L, 190mg/L, 180mg/L, 170mg/L, 160mg/L, 150mg/L, 140mg/L, 130mg/L, 120mg/L, 110mg/L, 100mg/L, 90mg/L, 80mg/L, 70mg/L, 60mg/L or 50mg/L. In one embodiment, the methods of the present invention reduce LDL levels to less than 160 mg/L, 150 mg/L, 140 mg/L, 130 mg/L, 120 mg/L, 110 mg/L, 100 mg/L, 90 mg/L, 80 mg/L, 70 mg/L, 60 mg/L, or 50 mg/L. In one embodiment, the methods of the present invention reduce LDL levels to less than 130 mg/L, 120 mg/L, 110 mg/L, 100 mg/L, 90 mg/L, 80 mg/L, 70 mg/L, 60 mg/L, or 50 mg/L. In one embodiment, the methods of the present invention reduce LDL levels to less than 100 mg/L, 90 mg/L, 80 mg/L, 70 mg/L, 60 mg/L, or 50 mg/L. In one embodiment, the methods of the present invention reduce LDL levels to less than 70 mg/L, 60 mg/L, or 50 mg/L.

In one embodiment, the lipid is a triglyceride. In one embodiment, compared to a control subject (for example, the subject of the composition of the present invention is not given), the method of the present invention reduces triglyceride levels by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In one embodiment, compared to healthy subjects (for example, the individuality without disease or illness (as described herein)), the subject has the triglyceride levels of elevation. In one embodiment, the method of the present invention reduces triglyceride levels to below 800mg/L, 700mg/L, 600mg/L, 500mg/L, 400mg/L, 300mg/L, 200mg/L, 190mg/L, 180mg/L, 170mg/L, 160mg/L, 150mg/L, 140mg/L, 130mg/L, 120mg/L, 110mg/L or 100mg/L. In one embodiment, the methods of the present invention reduce triglyceride levels to less than 200 mg/L, 190 mg/L, 180 mg/L, 170 mg/L, 160 mg/L, 150 mg/L, 140 mg/L, 130 mg/L, 120 mg/L, 110 mg/L, or 100 mg/L. In one embodiment, the methods of the present invention reduce triglyceride levels to less than 150 mg/L, 140 mg/L, 130 mg/L, 120 mg/L, 110 mg/L, or 100 mg/L.

The present invention also provides a method for reducing the amount of bilirubin and/or one or more liver enzymes, which comprises administering a therapeutically effective amount of the pharmaceutical composition of the present application to a subject in need thereof.

In one embodiment, compared to a control subject (e.g., a subject not given a composition of the present invention), the method of the present application reduces the amount of bilirubin by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In one embodiment, compared to a healthy subject (e.g., an individual without a disease or condition (e.g., those described herein), the subject has elevated bilirubin levels. In one embodiment, the method of the present application reduces the level of bilirubin to a normal level (e.g., similar to the level of bilirubin in an individual without a disease or condition (e.g., those described herein). In further embodiments, the method of the present application reduces the level of bilirubin to below 10 mg/L, 9 mg/L, 8 mg/L, 7 mg/L, 6 mg/L, 5 mg/L, 4 mg/L, 3 mg/L, 2 mg/L, 1.5 mg/L, 1.2 mg/L or 1 mg/L. In further embodiments, the methods of the present application reduce bilirubin levels to below 2 mg/L, 1.5 mg/L, 1.2 mg/L, or 1 mg/L.

In one embodiment, liver enzyme is selected from lower group, and this group is made up of the following: alkaline phosphatase (ALP, AP or AlkPhos), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), lactate dehydrogenase (LDH) and 5 ' nucleotidase.In one embodiment, compared to control subject (for example, the subject of the composition of the present invention is not given), the method of the application reduces the amount of one or more liver enzymes by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.In one embodiment, compared to healthy subject (for example, the individuality without disease or illness (as described herein)), this subject has one or more liver enzyme levels that increase.In one embodiment, the method of the application reduces the level of one or more liver enzymes (for example, ALP, ALT, AST, GGT, LDH and 5 ' nucleotidase) to normal level (for example, similar to the level of liver enzyme in the individuality without disease or illness (as described herein)).

In further embodiments, the methods of the present application reduce the level of ALP to below 500 IU/L (international units/liter), 400 IU/L, 300 IU/L, 200 IU/L, 180 IU/L, 160 IU/L, or 150 IU/L. In further embodiments, the methods of the present application reduce the level of ALP to from about 40 IU/L to about 150 IU/L.

In further embodiments, the methods of the present application reduce ALT levels to below 200 IU/L (international units/liter), 150 IU/L, 100 IU/L, 80 IU/L, 60 IU/L, or 50 IU/L. In further embodiments, the methods of the present application reduce ALT levels to from about 5 IU/L to about 50 IU/L.

In other embodiments, the methods of the present application reduce the level of AST to below 200 IU/L (international units/liter), 150 IU/L, 100 IU/L, 80 IU/L, 60 IU/L, 50 IU/L, or 40 IU/L. In other embodiments, the methods of the present application reduce the level of AST to from about 10 IU/L to about 50 IU/L.

In further embodiments, the methods of the present application reduce the level of GGT to below 200 IU/L (international units/liter), 150 IU/L, 100 IU/L, 90 IU/L, 80 IU/L, 70 IU/L, or 60 IU/L. In further embodiments, the methods of the present application reduce the level of GGT to from about 15 IU/L to about 50 IU/L or from about 5 IU/L to about 30 IU/L.

In other embodiments, the methods of the present application reduce the level of LDH to below 500 IU/L (international units/liter), 400 IU/L, 300 IU/L, 200 IU/L, 180 IU/L, 160 IU/L, 150 IU/L, 140 IU/L, or 130 IU/L. In other embodiments, the methods of the present application reduce the level of LDH to from about 120 IU/L to about 220 IU/L.

In further embodiments, the methods of the present application reduce the level of 5' nucleotidase to less than 50 IU/L (International Units/L), 40 IU/L, 30 IU/L, 20 IU/L, 18 IU/L, 17 IU/L, 16 IU/L, 15 IU/L, 14 IU/L, 13 IU/L, 12 IU/L, 11 IU/L, 10 IU/L, 9 IU/L, 8 IU/L, 7 IU/L, 6 IU/L, or 5 IU/L. In further embodiments, the methods of the present application reduce the level of 5' nucleotidase to from about 2 IU/L to about 15 IU/L.

In one embodiment, the methods of the present invention comprise administering to a subject in need thereof an effective amount of a first compound that is a FXR agonist, in combination with at least one PPAR-α agonist, PPAR-δ agonist, and/or PPAR-α and δ dual agonist, and optionally one or more pharmaceutically acceptable carriers. In another embodiment, the method comprises administering to a subject in need thereof an effective amount of a first compound in combination with at least one PPAR-α agonist, PPAR-δ agonist, and/or PPAR-α and δ dual agonist, wherein the first compound is a compound described herein (e.g., a compound of Formula A, I, IA, II, or IIA, or Compounds 1, 2, 3, or 4), or a pharmaceutically acceptable salt or amino acid conjugate thereof.

In one embodiment, the method of the present invention comprises administering to a subject in need thereof an effective amount of a first compound that is an FXR agonist in combination with at least one fibrate, and optionally one or more pharmaceutically acceptable carriers. In another embodiment, the method comprises administering to a subject in need thereof an effective amount of a first compound in combination with at least one fibrate, wherein the first compound is a compound described herein (e.g., a compound of Formula A, I, IA, II, or IIA, or Compound 1, 2, 3, or 4) or a pharmaceutically acceptable salt or amino acid conjugate thereof.

In one embodiment, the method of the present invention comprises administering to a subject in need thereof an effective amount of a first compound that is a FXR agonist in combination with at least one statin, and optionally one or more pharmaceutically acceptable carriers. In another embodiment, the method comprises administering to a subject in need thereof an effective amount of a first compound in combination with at least one statin, wherein the first compound is a compound described herein (e.g., a compound of Formula A, I, IA, II, or IIA, or Compounds 1, 2, 3, or 4), or a pharmaceutically acceptable salt or amino acid conjugate thereof.

In one embodiment, the methods of the present invention comprise administering to a subject in need thereof an effective amount of a first compound that is a FXR agonist, in combination with at least one PPAR-α agonist, PPAR-δ agonist, and/or PPAR-α and δ dual agonist, at least one statin, and optionally one or more pharmaceutically acceptable carriers. In another embodiment, the method comprises administering to a subject in need thereof an effective amount of a first compound in combination with at least one PPAR-α agonist, PPAR-δ agonist, and/or PPAR-α and δ dual agonist, at least one statin, wherein the first compound is a compound described herein (e.g., a compound of Formula A, I, IA, II, or IIA, or Compounds 1, 2, 3, or 4), or a pharmaceutically acceptable salt or amino acid conjugate thereof.

In one embodiment, the method of the present invention comprises administering to a subject in need thereof an effective amount of a first compound that is an FXR agonist in combination with at least one fibrate, at least one statin, and optionally one or more pharmaceutically acceptable carriers. In another embodiment, the method comprises administering to a subject in need thereof an effective amount of a first compound in combination with at least one fibrate, at least one statin, wherein the first compound is a compound described herein (e.g., a compound of Formula A, I, IA, II, or IIA, or Compound 1, 2, 3, or 4) or a pharmaceutically acceptable salt or amino acid conjugate thereof.

In one embodiment, the subject is a mammal. In one embodiment, the mammal is a human.

In one embodiment, the first compound and one or more PPAR-α agonists, one or more PPAR-δ agonists, one or more PPAR-α and δ or PPAR-α and γ dual agonists, one or more fibrates or one or more statins are administered in a two-way combination, i.e., in addition to the first compound and one or more PPAR-α agonists, one or more PPAR-δ agonists, one or more PPAR-α and δ or PPAR-α and γ dual agonists, one or more fibrates or one or more statins, there is no other therapeutic agent. In another embodiment, the first compound and one or more fibrates are administered in a two-way combination, i.e., in addition to the first compound and one or more fibrates, there is no other therapeutic agent. In another embodiment, the first compound and one or more statins are administered in a two-way combination, i.e., in addition to the first compound and one or more statins, there is no other therapeutic agent.

In another embodiment, the first compound and the one or more PPAR-alpha agonists, one or more PPAR-delta agonists, one or more PPAR-alpha and delta or PPAR-alpha and gamma dual agonists, or one or more fibrates are administered in a three-way combination with a statin. In a further embodiment, the first compound and one or more fibrates are administered in a three-way combination with a statin.

The first compound, together with one or more PPAR-alpha agonists, one or more PPAR-delta agonists, one or more PPAR-alpha and delta or PPAR-alpha and gamma dual agonists, or one or more fibrates and/or one or more statins, can achieve far-reaching synergistic effects, such as in severe mixed hyperlipidemic states and those resistant to individual therapy and in the coordinated reduction on one or more liver enzyme levels. Therefore, for very difficult to control hyperlipidemia, the combination of the first compound, PPAR-alpha agonists, PPAR-delta agonists, PPAR-alpha and delta or PPAR-alpha and gamma dual agonists, or fibrates and/or statins is advantageous. Such a combination of the first compound, fibrates, and/or statins is provided in a single pharmaceutical composition (such as in a single capsule form) with a pharmaceutically acceptable carrier designed to improve compliance and therefore improve effectiveness and can be particularly advantageous. Therefore, the present invention further provides a pharmaceutical composition comprising an effective amount of a first compound, an effective amount of at least one PPAR-α agonist, a PPAR-δ agonist, or a PPAR-α and δ or PPAR-α and γ dual agonist, and an effective amount of at least one statin, together with one or more pharmaceutically acceptable carriers, diluents, adjuvants or vehicles. In one embodiment, the present invention further provides a pharmaceutical composition comprising an effective amount of a first compound, an effective amount of at least one fibrate, and an effective amount of at least one statin, together with one or more pharmaceutically acceptable carriers, diluents, adjuvants or vehicles.

In one embodiment, the first compound and one or more PPAR-α agonists, one or more PPAR-δ agonists, one or more PPAR-α and δ or PPAR-α and γ dual agonists, or one or more fibrates are administered simultaneously. For example, the first compound and one or more PPAR-α agonists, one or more PPAR-δ agonists, one or more PPAR-α and δ or PPAR-α and γ dual agonists, or one or more fibrates are administered as a single pharmaceutical composition together with a pharmaceutically acceptable carrier. In another embodiment, the first compound and one or more PPAR-α agonists, one or more PPAR-δ agonists, one or more PPAR-α and δ or PPAR-α and γ dual agonists, or one or more fibrates are administered sequentially. For example, the first compound is administered before or after one or more PPAR-α agonists, one or more PPAR-δ agonists, one or more PPAR-α and δ or PPAR-α and γ dual agonists, or one or more fibrates.

In one embodiment, the first compound and the statin are administered simultaneously. For example, the first compound and the statin are administered as a single pharmaceutical composition together with a pharmaceutically acceptable carrier. In another embodiment, the first compound and the statin are administered sequentially. For example, the first compound is administered before or after the statin.

In one embodiment, the first compound is administered at a first dose for a first period of time, followed by administration of the first compound at a second dose for a second period of time. In one embodiment, the first compound or a pharmaceutically acceptable salt or amino acid conjugate thereof is administered at 0.1-1500 mg, 0.2-1200 mg, 0.3-1000 mg, 0.4-800 mg, 0.5-600 mg, 0.6-500 mg, 0.7-400 mg, 0.8-300 mg, 1-200 mg, 1-100 mg, 1-50 mg, 1-30 mg, 4-26 mg, or 5-25 mg. In one embodiment, the first compound is administered orally at a daily total amount of 0.1-1500mg, 0.2-1200mg, 0.3-1000mg, 0.4-800mg, 0.5-600mg, 0.6-500mg, 0.7-400mg, 0.8-300mg, 1-200mg, 1-100mg, 1-50mg, 1-30mg, 4-26mg or 5-25mg. In one embodiment, the total amount is administered orally once daily. In one embodiment, the first dose is different from the second dose. In another embodiment, the first dose is lower than the second dose. In another embodiment, the first dose is higher than the second dose. In one embodiment, the first dose is about 5mg (e.g., from 4.8mg to 5.2mg), and the second dose is about 10mg (e.g., from 9.8mg to 10.2mg). In one embodiment, the first time period is about 6 months. In one embodiment, the second time period is about 6 months.

In one embodiment, the pharmaceutical composition is administered orally, parenterally, or topically. In another embodiment, the pharmaceutical composition is administered orally.

The compositions according to the present invention will typically contain sufficient first compound or a pharmaceutically acceptable salt or amino acid conjugate thereof, one or more PPAR-α agonists, one or more PPAR-δ agonists, one or more PPAR-α and δ or PPAR-α and γ dual agonists, or one or more fibrates, and/or one or more statins to allow the desired daily dose of each to be administered to a subject in need thereof in a single unit dosage form (e.g., a tablet or capsule) or in two or more unit dosage forms administered simultaneously or at intervals during the day.

The present invention also provides a pharmaceutical composition wherein the first compound and one or more PPAR-α agonists, one or more PPAR-δ agonists, one or more PPAR-α and δ or PPAR-α and γ dual agonists, or one or more fibrates are administered in combination with UDCA. In one aspect, UDCA is administered in a three-way combination. In another aspect, a two-way combination of the first compound and one or more PPAR-α agonists, one or more PPAR-δ agonists, one or more PPAR-α and δ or PPAR-α and γ dual agonists, or one or more fibrates is administered in place of UDCA to a patient who has an inadequate therapeutic response to UDCA alone for the treatment or prevention of a disease or condition.

In the methods of the present invention, the active substances may be administered in a single daily dose or in two, three, four or more identical or different divided doses per day, and these doses may be administered simultaneously or at different times during the day. Typically, the active substances will be administered simultaneously, more typically in a single combined dosage form.

In one aspect, the first compound, one or more PPAR-alpha agonists, one or more PPAR-delta agonists, one or more PPAR-alpha and delta or PPAR-alpha and gamma dual agonists, or one or more fibrates, and/or one or more statins are administered at a dose substantially the same as the dose they are administered in their respective monotherapy. In one aspect, the first compound is administered at a dose less than (e.g., less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%) its monotherapy dose. In one aspect, the one or more PPAR-alpha agonists, one or more PPAR-delta agonists, one or more PPAR-alpha and delta or PPAR-alpha and gamma dual agonists, or one or more fibrates are administered at a dose less than (e.g., less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%) its monotherapy dose. In one aspect, the first compound and one or more PPAR-alpha agonists, one or more PPAR-delta agonists, one or more PPAR-alpha and delta or PPAR-alpha and gamma dual agonists, or one or more fibrates are administered at a dose less than (e.g., less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%) their respective monotherapy doses. In one aspect, the one or more statins are administered at a dose less than (e.g., less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%) their respective monotherapy doses. In one aspect, the first compound and one or more statins are administered at a dose less than (e.g., less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%) their respective monotherapy doses. In one aspect, the first compound, one or more PPAR-alpha agonists, one or more PPAR-delta agonists, one or more PPAR-alpha and delta or PPAR-alpha and gamma dual agonists, or one or more fibrates, and/or one or more statins are administered at a dose that is less than (e.g., less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%) their respective monotherapy doses.

The pharmaceutical compositions of the present invention may be in any convenient form for oral administration, such as tablets, capsules, powders, lozenges, pills, troches, elixirs, lyophilized powders, solutions, granules, suspensions, emulsions, syrups, or tinctures. Sustained-release, modified-release, or delayed-release forms may also be prepared, for example, in the form of coated granules, multilayer tablets, capsules within capsules, tablets within capsules, or microgranules.

Solid forms for oral administration can contain pharmaceutically acceptable binders, sweeteners, disintegrants, diluents, flavorings, coatings, preservatives, lubricants, and/or delay agents. Suitable binders include gum arabic, gelatin, corn starch, tragacanth gum, sodium alginate, carboxymethyl cellulose, or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame, or saccharin. Suitable disintegrants include corn starch, methylcellulose, polyvinyl pyrrolidone, xanthan gum, bentonite, alginic acid, or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate, or dicalcium phosphate. Suitable flavorings include peppermint oil, wintergreen oil, cherry, orange, or raspberry flavorings. Suitable coatings include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac, or gluten. Suitable preservatives include sodium benzoate, vitamin E, α-tocopherol, ascorbic acid, methylparaben, propylparaben or sodium bisulfite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable delay agents include glyceryl monostearate or glyceryl distearate.

In addition to the above-mentioned medicaments, liquid forms for oral administration can also contain a liquid carrier. Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, groundnut oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropyl alcohol, glycerol, fatty alcohol, triglyceride or a mixture thereof.

Suspensions for oral administration may further include dispersants and/or suspending agents. Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, sodium alginate, or cetyl alcohol. Suitable dispersants include lecithin; polyoxyethylene esters of fatty acids such as stearic acid; polyoxyethylene sorbitan mono- or di-oleate, stearate, or laurate; polyoxyethylene sorbitan mono- or di-oleate, stearate, or laurate, etc.

Emulsions for oral administration may further comprise one or more emulsifiers. Suitable emulsifiers include dispersants as exemplified above or natural gums such as acacia or tragacanth.

The pharmaceutical composition of the present invention can be prepared by blending, grinding, homogenizing, suspending, dissolving, emulsifying, dispersing and/or mixing the first compound or its pharmaceutically acceptable salt or amino acid conjugate and at least one lipid-lowering agent (e.g., fibrate), and optionally one or more statins, together with selected one or more excipients, one or more carriers, one or more adjuvants and/or one or more diluents. A type of pharmaceutical composition in the form of tablets or capsules of the present invention can be prepared in the following manner: (a) preparing a first tablet, the first tablet comprising at least one of the active substances selected from the first compound or its pharmaceutically acceptable salt or amino acid conjugate and at least one lipid-lowering agent, together with any desired one or more excipients, one or more carriers, one or more adjuvants and/or one or more diluents; and (b) preparing a second tablet or capsule, wherein the second tablet or capsule comprises the remaining one or more active substances and the first tablet. One type of pharmaceutical composition of the present invention in the form of a capsule can be prepared by: (a) preparing a first capsule comprising at least one of the active substances selected from the first compound or its pharmaceutically acceptable salt or amino acid conjugate, and one or more lipid-lowering agents, together with any desired excipient(s), carrier(s), adjuvant(s) and/or diluent(s); and (b) preparing a second capsule, wherein the second capsule comprises the remaining active substance(s) and the first capsule. Another type of pharmaceutical composition of the present invention in the form of a tablet can be prepared by: (a) preparing a capsule comprising at least one of the active substances selected from the first compound or its pharmaceutically acceptable salt or amino acid conjugate, and at least one lipid-lowering agent, together with any desired excipient(s), carrier(s), adjuvant(s) and/or diluent(s); and (b) preparing a tablet, wherein the tablet comprises the remaining active substance(s) and the capsule.

In an embodiment, the one or more PPAR-α agonists, one or more PPAR-δ agonists, one or more PPAR-α and δ or PPAR-α and γ dual agonists, or one or more fibrates, and/or one or more statins are used as immediate release tablets or sustained release tablets. It is particularly effective when provided in a sustained release tablet. Sustained release tablets of various lipid-lowering agents are commercially available. It is preferred that the tablet be in a sustained release form for prolonged action.

In another embodiment, the pharmaceutical composition of the present invention comprises a capsule containing one or more PPAR-α agonists, one or more PPAR-δ agonists, one or more PPAR-α and δ or PPAR-α and γ dual agonists, or one or more fibrates, and/or one or more statins, wherein the capsule contains the first compound or a pharmaceutically acceptable salt or amino acid conjugate thereof. Typically, in this form, the one or more PPAR-α agonists, one or more PPAR-δ agonists, one or more PPAR-α and δ or PPAR-α and γ dual agonists, or one or more fibrates, and/or one or more statins are presented in an immediate release form. In this event, the composition is usually administered three times a day. Another mode of administration is to provide the composition containing the one or more PPAR-α agonists, one or more PPAR-δ agonists, one or more PPAR-α and δ or PPAR-α and γ dual agonists, or one or more fibrates, and/or one or more statins in a sustained release form or a non-sustained release form as described above twice a day, wherein the daily amount of the composition administered contains a sufficient amount of active substance to provide the patient with the desired daily dose.

In one embodiment, the pharmaceutical composition of the present invention is a dosage form comprising a total daily amount of 0.1-1500 mg, 0.2-1200 mg, 0.3-1000 mg, 0.4-800 mg, 0.5-600 mg, 0.6-500 mg, 0.7-400 mg, 0.8-300 mg, 1-200 mg, 1-100 mg, 1-50 mg, 1-30 mg, 4-26 mg, or 5-25 mg of the first compound or a pharmaceutically acceptable salt or amino acid conjugate thereof. In one embodiment, the total amount is administered orally once daily.

In one embodiment, the pharmaceutical composition of the present invention is a dosage form comprising a total daily amount of 10-1000 mg, 20-800 mg, 50-500 mg, 80-400 mg, or 100-300 mg, more typically about 200 mg of a PPAR-α agonist, a PPAR-δ agonist, a PPAR-α and δ or PPAR-α and γ dual agonist, or a fibrate, and/or a statin. In one embodiment, the total amount is administered orally once daily. In one embodiment, the pharmaceutical composition of the present invention is a dosage form comprising a statin in an amount of 5-1000 mg, 10-800 mg, 20-500 mg, 30-400 mg, or 40-200 mg.

In an embodiment, the composition of the invention is a dosage form comprising an amount of a PPAR-alpha agonist, a PPAR-delta agonist, a PPAR-alpha and delta or PPAR-alpha and gamma dual agonist, or a fibrate, and/or a statin in an amount of 10-1000 mg, 20-800 mg, 50-500 mg, 80-400 mg, or 100-300 mg, more typically about 200 mg, contained in a capsule containing an amount of the first compound in an amount of 0.1-1500 mg, 0.2-1200 mg, 0.3-1000 mg, 0.4-800 mg, 0.5-600 mg, 0.6-500 mg, 0.7-400 mg, 0.8-300 mg, 1-200 mg, 1-100 mg, 1-50 mg, 1-30 mg, 4-26 mg, or 5-25 mg. In one embodiment, the PPAR-alpha agonist, PPAR-delta agonist, PPAR-alpha and delta or PPAR-alpha and gamma dual agonist, or fibrate, and/or statin is in sustained release form.

In an embodiment, the composition of the present invention is a dosage form of a sustained-release tablet containing 10-1000 mg, 20-800 mg, 50-500 mg, 80-400 mg, or 100-300 mg, more typically about 200 mg of bezafibrate contained in a capsule containing 0.1-1500 mg, 0.2-1200 mg, 0.3-1000 mg, 0.4-800 mg, 0.5-600 mg, 0.6-500 mg, 0.7-400 mg, 0.8-300 mg, 1-200 mg, 1-100 mg, 1-50 mg, 1-30 mg, 4-26 mg, or 5-25 mg of the first compound. In this way, a patient administered this dosage form receives a sustained-release tablet of bezafibrate, which is delivered to the distal cavity as the capsule breaks open and releases the first compound.

Pharmaceutical composition of the present invention can be used by patient for life, prolongs life span and delays liver transplantation.The reduction of hyperlipidemia and liver enzymes guarantees the reduction of the development of related vascular diseases.The first compound and lipid-lowering agent (such as benzafate and/or statin) all have very little long-term side effect characteristics (except bezafibrate), so this combination may be the treatment selection of primary biliary cirrhosis (PBC) and resistance primary biliary cirrhosis (PBC) with hyperlipidemia.Due to the simplified administration provided by the present invention, the combination therapy of the present invention may depend on the patient's weight and clinical response to use with increased dosage.

The composition comprising the first compound or its pharmaceutically acceptable salt or amino acid conjugate, PPAR-α agonist, PPAR-δ agonist, PPAR-α and δ or PPAR-α and γ dual agonist or fibrate and/or statin of the present invention can be provided as three active substances in a single capsule. In one form of such a composition, statin can be mixed with the first compound in an inner capsule, and the inner capsule is surrounded by the PPAR-α agonist, PPAR-δ agonist, PPAR-α and δ or PPAR-α and γ dual agonist or fibrate contained in the outer capsule. The position in the capsule can be reversed. That is, the mixture of statin and the first compound can be contained in the outer capsule, and the PPAR-α agonist, PPAR-δ agonist, PPAR-α and δ or PPAR-α and γ dual agonist or fibrate can be contained in the inner capsule. If the amount of statin to be administered is relatively large, this arrangement will be particularly satisfactory. Other combinations for administering the three active substance combinations are possible.

The first compound disclosed herein can be prepared by conventional methods (e.g., those described in U.S. Publication No. 2009/0062526, U.S. Pat. No. 7,138,390, and WO 2006/122977), such as by a 6-step synthesis followed by a purification step, to produce highly pure Compound 1 (obeticholic acid or OCA), as shown in Scheme 1 below.

Solution 1

The above method is described in WO 2013/192097, the contents of which are incorporated herein by reference in their entirety. The method is a 6-step synthesis followed by a purification step. Step 1 is the esterification of the C-24 carboxylic acid of 7-ketolithocholic acid (KLCA) to produce the methyl ester compound a. Step 2 is the formation of an enol silyl ether from compound 1 to produce compound c. Step 3 is the aldol condensation reaction of the enol silyl ether compound c with acetaldehyde to produce compound d. Step 4 is the saponification of compound d to produce compound e. Step 5 is the hydrogenation of compound e to produce compound f. Step 6 is the selective reduction of the 7-keto group of compound f to produce crystalline compound 1. Step 7 is the conversion of the crystalline compound into amorphous compound 1 (obeticholic acid form 1 or OCA form 1).

Alternatively, the first compound disclosed herein can be prepared by conventional methods (e.g., those described in U.S. Patent No. 7,932,244) or via a process as shown in Scheme 2 (and disclosed in WO 2014/066819). Scheme 2 can be used to prepare compounds 2, 3, or 4 disclosed herein.

Step 1 is the esterification of a compound of formula II to obtain a compound of formula III. Step 2 is the reaction of forming a compound of formula IV from a compound of formula III. Step 3 is the protection of the hydroxyl group at the C3 position of the compound of formula IV to provide a compound of formula V. Step 4 is the oxidative cleavage of the compound of formula V to give a compound of formula VI. Step 5 is the reduction of the compound of formula VI to provide a compound of formula VII. Step 6 is the sulfonation of the compound of formula VII to give a salt of formula I-Na. The salt of formula I-Na can be converted into its free base form (i.e., a compound of formula I) or other salt forms (e.g., a salt of formula I-(Et) ₃ NH)).

definition

For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

As used herein, the term "fibrate" refers to any of the fibric acid derivatives and pharmaceutically active derivatives of 2-phenoxy-2-methylpropionic acid that can be used in the methods described herein. Examples of fibrates include, but are not limited to, fenofibrate, bezafibrate, benzclofibrate, binifibrate, ciprofibrate, clinofibrate, clofibrate, clofibric acid, etofibrate, gemfibrozil, nicofibrate, pirfibrate, clonifibrate, simfibrate, clofibrate, tocofibrate, prafibride, and the like. Examples of beta-benzyl alcohol are also described in U.S. Patent Nos. 3,781,328, 3,948,973, 3,869,477, 3,716,583, 3,262,580, 3,723,446, 4,058,552, 3,674,836, 3,369,025, 3,984,413, 3,971,798, 6,384,062, 7,119,198, and 7,259,186; U.S. Publication No. 20090131395; WO 2008/039829; Belgian Patent No. 884722; British Patent No. 860303; and European Patent Application Publication No. EP 0607536, the entire disclosure of each of which is incorporated herein by reference.

Peroxisome proliferator-activated receptor alpha (PPAR-α), also known as NR1C1 (nuclear receptor subfamily 1, group C, member 1), is a nuclear receptor protein. PPAR-α agonists bind to and activate PPAR-α. Examples of PPAR-α agonists include, but are not limited to, fibrates, such as those described herein.

Peroxisome proliferator-activated receptor delta (PPAR-δ), also known as NR1C2 (nuclear receptor subfamily 1, group C, member 2), is a nuclear receptor protein. PPAR-δ agonists bind to and activate PPAR-δ. Examples of PPAR-δ agonists include, but are not limited to, {4-[({4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl}methyl)sulfanyl]-2-methylphenoxy}acetic acid (also known in the art as GW501516, GW1516, and Endurabol), {2-methyl-4-[5-methyl-2-(4-trifluoromethyl-phenyl)-2H-[1,2,3]triazol-4-ylmethylsulfanyl]-phenoxy}-acetic acid, and [4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphenoxy]-acetic acid.

PPAR-α and δ or PPAR-α and γ dual agonists bind to and activate both PPAR-α and PPAR-δ, or both PPAR-α and PPAR-γ. Examples of PPAR-α and δ dual agonists include, but are not limited to, 2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1(E)-propenyl]phenoxy]-2-methylpropanoic acid (also known as GFT505). Examples of PPAR-α and γ dual agonists include, but are not limited to, aleglitazar ((2S)-2-methoxy-3-[4-[2-(5-methyl-2-phenyl-4-oxazolyl)ethoxy]-7-benzophenylthio]propionic acid, CAS No. 475479-34-6), moglitazole (N-[(4-methoxyphenoxy)carbonyl]-N-{4-[2-(5-methyl-2-phenyl-1,3-oxazol-4-yl)ethoxy]benzyl}glycine, CAS No. 331741-94-7), tesaglitazar ((2S)-2-ethoxy-3-[4-[2-(4-methylsulfonyloxyphenyl)ethoxy]phenyl]propanoic acid, CAS No. 251565-85-2) and saroglitazar ((2S)-2-ethoxy-3-[4-(2-{2-methyl-5-[4-(methylsulfanyl)phenyl]-1H-pyrrol-1-yl}ethoxy)phenyl]propanoic acid, CAS No. 495399-09-2).

As used herein, the term "FXR agonist" refers to any compound that activates FXR. In one aspect, the FXR agonist achieves at least 50% activation of FXR relative to CDCA, and appropriate positive controls for these assays are described in WO 2000/037077. In another aspect, the FXR agonist achieves 100% activation of FXR in a scintillation proximity assay or HTRF assay as described in WO 2000/037077. Examples of FXR agonists include, but are not limited to, those described in U.S. 7,138,390; 7,932,244; 20120283234; 20120232116; 20120053163; 20110105475; 20100210660; 20100184809; 20100172870; 20100152166; 20100069367; 20100063018; 20100022498; 20090270460; 20090215748; 20090163474; 20090093524; 20080300235; 20080299118; 20080182832; 20080039435; 20070142340; 20060069070; 20050080064; 20040176426; 20030130296; 20030109467; 20030003520; 20020132223; and 20020120137.

As used herein, the term "obeticholic acid" or "OCA" refers to a compound having the following chemical structure:

Obeticholic acid is also known as obeticholic acid form 1, INT-747, 3α, 7α-dihydroxy-6α-ethyl-5β-cholan-24-oic acid, 6α-ethyl-chenodeoxycholic acid, 6-ethyl-CDCA, 6ECDCA, cholan-24-oic acid, 6-ethyl-3,7-dihydroxy-(3α,5β,6α,7α), and can be prepared by the methods described in U.S. Publication No. 2009/0062526A1, U.S. Patent No. 7,138,390, and WO2006/122977. The CAS registry number of obeticholic acid is 459789-99-2.

As used herein, the term "crystalline obeticholic acid" refers to any crystalline form of a compound having the following chemical structure:

Crystalline obeticholic acid means that the compound crystallizes into a specific crystal packing arrangement in three spatial dimensions or the compound has an outer surface plane. The crystalline form of obeticholic acid (or a pharmaceutically acceptable salt thereof) can crystallize into different crystal packing arrangements, all of which have the same elemental composition of obeticholic acid. Different crystal forms typically have different X-ray diffraction patterns, infrared spectra, melting points, density hardness, crystal shape, optical and electrical properties, stability, and solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors may cause one crystal form to dominate. Crystals of obeticholic acid can be prepared by crystallization under different conditions (e.g., different solvents, temperatures, etc.). Examples of crystalline forms of OCA are described in U.S. Patent No. 9,238,673.

The term "first compound" means a compound of Formula A, I, IA, II or IIA, or Compound 1, 2, 3 or 4, or a pharmaceutically acceptable salt or amino acid conjugate thereof. Whenever the term is used in the context of the present invention, it should be understood to refer to the free base, isotopically labeled compound, crystalline compound, or its corresponding pharmaceutically acceptable salt or amino acid conjugate, provided that this is possible and/or appropriate under the circumstances.

As used herein, the term "amino acid conjugate" refers to a conjugate of a first compound of the invention (e.g., a compound of Formula A) with any suitable amino acid. For example, such a suitable amino acid conjugate of a compound of Formula A would have the added advantage of enhanced integrity in bile or intestinal fluid. Suitable amino acids include, but are not limited to, glycine and taurine. Thus, the present invention encompasses glycine and taurine conjugates of a first compound of the invention (e.g., Compound 1).

The term "statin" is synonymous with the terms "3-hydroxy-3-methylglutaryl-CoA reductase inhibitor" and "HMG-CoA reductase inhibitor." These terms are used interchangeably herein. As the synonyms indicate, statins are inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase and are therefore effective in lowering plasma cholesterol levels and are therefore used to treat or prevent cardiovascular disease. Statins and their pharmaceutically acceptable salts are particularly effective in lowering low-density lipoprotein cholesterol (LDL-C) levels in mammals, especially humans. Structurally, statins or their derivatives have a common 4-hydroxy-6-oxo-2H-pyran system, which can also be in the form of a dihydroxy acid that interacts with the active site of HMG-CoA reductase and a lipophilic moiety that exists, in particular, as a polysubstituted hexahydronaphthalene system, but can also be replaced by polysubstituted heteroaromatic systems, as in atorvastatin or fluvastatin. Statins suitable for use herein include, but are not limited to, simvastatin, fluvastatin, pravastatin, rivastatin, mevastatin, atorvastatin, cerivastatin, lovastatin, pitavastatin, fludostatin, vilvastatin, darvastatin, rosuvastatin, dihydrocompactin and compatin, or pharmaceutically acceptable salts thereof.

The term "lipid-lowering agent" refers to any agent that can reduce the concentration of lipids (e.g., cholesterol, LDL, and triglycerides) in the circulation (e.g., in the blood). Lipid-lowering agents include, but are not limited to, (i) bile acid sequestrants, such as resins (e.g., cholestyramine, colestipol, colesevelam); (ii) cholesterol absorption inhibitors, which prevent cholesterol uptake (e.g., from the small intestine into the circulation), such as ezetimibe (i.e., (3R,4S)-1-(4-fluorophenyl)-3-[(3S)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)azetidin-2-one) and (3R,4S)-1,4-bis(4-methoxyphenyl)-3-(3-phenylpropyl)-2-azetidin-1-one); (iii) omega-3 fatty acid ethyl esters, including free fatty acid derivatives (e.g., Vascepa ^™ , Epadel, Epanova ^™ ) or marine-derived ω-3 polyunsaturated fatty acids (PUFAs); (iv) PCSK9 inhibitors; (v) niacin; (vi) plant sterols (e.g., plant sterols and stanols), such as β-sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, β-sitostanol, campesterol, stigmasterol, cycloartenol, and lupeol; (vii) inhibitors of CETP (cholesterol ester transfer protein), such as anacetrapib, evacetrapib, torcetrapib, and dalcetrapib; (viii) squalene synthase inhibitors; (ix) antisense oligonucleotides that affect the synthesis, degradation, absorption and metabolism of lipids (e.g., antisense oligonucleotides that bind to mRNA encoding apolipoprotein B or PCSK9) (e.g., Mipomersen (Kynamro)); (x) apoprotein-B inhibitors; (xi) inhibitors of microsomal triglyceride transfer protein (e.g., Lomitapide (Juxtapid)); and (xii) other compounds such as colesevelam, avasimib and implitapide.

"Treatment" includes any action that results in improvement of a condition, disease, disorder, etc., such as alleviation, reduction, regulation, or elimination. "Treating" or "treatment" of a disease state includes: inhibiting the disease state, i.e., arresting the development of the disease state or its clinical symptoms; or relieving the disease state, i.e., causing temporary or permanent regression of the disease state or its clinical symptoms.

"Preventing" a disease state includes preventing the development of clinical symptoms of the disease state in a subject who may be exposed to or susceptible to the disease state, but who does not yet experience or display symptoms of the disease state.

As used herein, the term "inhibiting" or "inhibition" refers to any detectable positive effect on the development or progression of a disease or condition. Such a positive effect can include delaying or preventing the onset of at least one symptom or sign of a disease or condition, alleviating or reversing the one or more symptoms or one or more signs, and slowing or preventing further worsening of the one or more symptoms or one or more signs.

"Disease state" means any disease, disorder, condition, symptom, or indication.

As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of the first compound (e.g., FXR activating ligand) or fibrate, or lipid-lowering agent, or statin that produces an acute or chronic therapeutic effect after appropriate dosage administration (alone or in combination). In one embodiment, an effective amount or therapeutically effective amount of the first compound (e.g., FXR activating ligand) produces an acute or chronic therapeutic effect after appropriate dosage administration in combination with at least one fibrate. This effect includes the prevention, correction, suppression, or reversal of symptoms, signs, and potential pathologies of the disease/disorder (e.g., fibrosis of the liver, kidney, or intestine) and related complications to any detectable extent. An "effective amount" or "therapeutically effective amount" will vary depending on the first compound, fibrate, lipid-lowering agent, statin, disease and its severity, and the age, weight, etc. of the subject to be treated.

The first compound of therapeutically effective amount can be formulated for administration to humans or non-human animals together with one or more fibrates and optionally one or more pharmaceutically acceptable carriers. Therefore, the pharmaceutical composition of the present invention can be administered, for example, via oral, parenteral or topical routes to provide an effective amount of the first compound and one or more fibrates. In alternative embodiments, the composition of the present invention can be used for coating or impregnating medical devices, such as stents.

As used herein, "pharmacological effect" encompasses an effect produced in a subject that achieves the intended purpose of a therapy. In one embodiment, a pharmacological effect is intended to prevent, alleviate, or reduce the primary indication of the treated subject. For example, a pharmacological effect would be a pharmacological effect that produces a prevention, alleviation, or reduction of the primary indication in the treated subject. In another embodiment, a pharmacological effect is intended to prevent, alleviate, or reduce the disorder or symptom of the primary indication of the treated subject. For example, a pharmacological effect would be a pharmacological effect that produces a prevention, alleviation, or reduction of the disorder or symptom of the treated subject.

It should be understood that unless otherwise stated, isomers arising from asymmetric carbon atoms (eg, all enantiomers and diastereomers) are included within the scope of the present invention. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis.

A "pharmaceutical composition" is a formulation containing a therapeutic agent (such as a first compound) and a lipid-lowering agent (such as a fibrate) in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk form or unit dosage form. It can be advantageous to formulate the composition in a dosage unit form that is easy to administer and achieves uniformity of dosage. As used herein, a unit dosage form refers to a physically discrete unit suitable as a single dose for a subject to be treated; each unit contains a predetermined amount of an active agent calculated to produce the desired therapeutic effect associated with the required pharmaceutical carrier. The specifications of the dosage unit form of the present invention are determined by and directly depend on the unique characteristics of the active agent and the specific therapeutic effect to be achieved, and the inherent limitations of such active agents formulated in the art for treating individuals.

The term "unit dosage forms" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with a suitable pharmaceutical excipient as described herein.

Unit dosage form is any one of various forms, including single pumps or vials on capsules, IV bags, tablets, aerosol inhalers, etc. The amount of the first compound or its pharmaceutically acceptable salt or amino acid conjugate in the unit dose of the composition is an effective amount and changes according to the specific treatment involved and/or one or more lipid-lowering agents for the treatment. It will be appreciated by those skilled in the art that it is sometimes necessary to make conventional changes to the dosage according to the patient's age and illness. The dosage will also depend on the route of administration. Various routes have been considered, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalation, buccal, sublingual, intrapleural, intrathecal, intranasal, etc. The dosage form for topical or transdermal administration of the compound of the present invention includes powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. In one embodiment, the first compound and/or lipid-lowering agent are mixed under aseptic conditions with a pharmaceutically acceptable carrier and with any preservative, buffer, or propellant required.

The term "flash release dosage form" refers to a formulation that is in a rapidly dispersing dosage form.

The term "immediate release" is defined as the release of a therapeutic agent (such as a first compound or a lipid-lowering agent) from a dosage form over a relatively short period of time (typically up to about 60 minutes). The term "modified release" is defined to include delayed release, extended release, and pulsatile release. The term "pulsatile release" is defined as a series of releases of a drug from a dosage form. The terms "sustained release" or "extended release" are defined as the continuous release of a therapeutic agent from a dosage form over an extended period of time.

"Subject" includes mammals, e.g., humans, companion animals (e.g., dogs, cats, birds, etc.), farm animals (e.g., cattle, sheep, pigs, horses, poultry, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, birds, etc.). In one embodiment, the subject is a human. In one aspect, the subject is female. In one aspect, the subject is male.

As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, carriers and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

"Pharmaceutically acceptable carrier or excipient" means a carrier or excipient that is generally safe, non-toxic, and not biologically or otherwise undesirable and is suitable for use in preparing pharmaceutical compositions, and includes excipients that are acceptable for veterinary use as well as human pharmaceutical use. As used in this specification and claims, "pharmaceutically acceptable excipient" includes both one and more than one such excipient.

While it is possible to administer the first compound directly without any formulation, the first compound may be administered in the form of a pharmaceutical formulation comprising a pharmaceutically acceptable excipient. Such formulations may be administered by various routes including oral, buccal, rectal, intranasal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal.

In one embodiment, the first compound can be administered transdermally. In order to administer transdermally, a transdermal delivery device ("patch") is required. Such transdermal patches can be used for continuous or intermittent infusion of the compound of the present invention in a controlled amount. The construction and use of transdermal patches for pharmaceutical agent delivery are well known in the art. See, for example, U.S. Patent number 5,023,252. Such patches can be made into pharmaceutical agents that are continuous, pulsed, or delivered on demand.

In one embodiment, the pharmaceutical composition of the present invention is suitable for buccal and/or sublingual or nasal administration. This embodiment provides for administration of the first compound in a manner that avoids gastric complications (e.g., first-pass metabolism through the gastric system and/or through the liver). This route of administration can also reduce adsorption time, thereby providing a faster onset of therapeutic benefit.

The first compound can be administered within a wide dosage range. For example, the daily dose is typically within the range of about 0.0001 to about 30 mg/kg body weight. In the treatment of adults, a range of about 0.1 to about 15 mg/kg/day in a single or divided dose can be used. In one embodiment, the formulation comprises about 0.1 mg to about 1500 mg of the first compound. In another embodiment, the formulation comprises about 1 mg to about 100 mg of the first compound. In another embodiment, the formulation comprises about 1 mg to about 50 mg of the first compound. In another embodiment, the formulation comprises about 1 mg to about 30 mg of the first compound. In another embodiment, the formulation comprises about 4 mg to about 26 mg of the first compound. In another embodiment, the formulation comprises about 5 mg to about 25 mg of the first compound. However, it should be understood that the amount of the first compound actually administered will be determined by the physician based on relevant circumstances (including the condition to be treated, the selected route of administration, the form of the first compound administered, the one or more lipid-lowering agents administered, the age, weight and response of the individual patient, and the severity of the patient's symptoms, etc.). Therefore, the above dosage ranges are not intended to limit the scope of the present invention in any way. In some cases, dosage levels below the lower limit of the aforesaid range may be sufficient, while in other cases still larger doses may be employed without causing any deleterious side effects, provided that such larger doses are first divided into several smaller doses for administration throughout the day.

"Fibrosis" refers to a condition involving the development of excess fibrous connective tissue (e.g., scar tissue) in a tissue or organ. This production of scar tissue can occur in response to infection, inflammation, or damage to an organ due to disease, trauma, chemical toxicity, etc. Fibrosis can develop in a variety of different tissues and organs, including the liver, kidneys, intestines, lungs, heart, etc.

As used herein, "cholestatic disorder" refers to any disease or disorder in which bile excretion from the liver is impaired or blocked, which can occur in the liver or in the bile duct. Intrahepatic cholestasis and extrahepatic cholestasis are two types of cholestatic disorders. Intrahepatic cholestasis (occurring inside the liver) is most commonly seen in primary biliary cirrhosis, primary sclerosing cholangitis, sepsis (systemic infection), acute alcoholic hepatitis, drug poisoning, total parenteral nutrition (intravenous supply), malignant tumors, cystic fibrosis, biliary atresia, and pregnancy. Extrahepatic cholestasis (occurring outside the liver) can be caused by stone formation in the bile duct tumors, strictures, cysts, diverticula, common bile duct, pancreatitis, pancreatic tumors or pseudocysts, and compression due to masses or tumors in nearby organs.

The clinical symptoms and signs of cholestatic disorders include: itching (pruritus), fatigue, jaundice skin or eyes, inability to digest certain foods, nausea, vomiting, white stools, dark yellow urine, and right upper quadrant pain. Patients suffering from cholestatic disorders can be diagnosed and followed up clinically based on a set of standard clinical laboratory tests, which include measurements of alkaline phosphatase, gamma-glutamyl transpeptidase (GGT), 5' nucleotidase, bilirubin, bile acid, and cholesterol levels in the patient's serum. Typically, if the serum levels of all three diagnostic markers (alkaline phosphatase, GGT, and 5' nucleotidase) are considered to be abnormally elevated, the patient is diagnosed as suffering from cholestatic disorders. The normal serum levels of these markers may depend on the test protocol and vary to a certain extent depending on the laboratory and procedure. Therefore, the doctor will be able to determine based on specific laboratory and test procedures what the blood levels for each abnormal increase in these markers are. For example, patients with cholestatic disorders typically have greater than about 125 IU/L of alkaline phosphatase, greater than about 65 IU/L of GGT, and greater than about 17 IU/L of 5' nucleotidase in their blood. Due to the variability in the levels of serum markers, cholestatic disorders can be diagnosed based on abnormal levels of these three markers in addition to at least one of the above symptoms, such as itching (pruritus).

The term "primary biliary cirrhosis," often abbreviated as PBC, is an autoimmune disease of the liver characterized by the slow, progressive destruction of the small bile ducts of the liver, with the intralobular ducts (ducts of Hering) being affected early in the disease. When these ducts are damaged, bile accumulates in the liver (cholestasis) and damages the tissue over time. This can lead to scarring, fibrosis, and cirrhosis. Primary biliary cirrhosis is characterized by destruction of the interlobular bile ducts. Histopathological findings in primary biliary cirrhosis include inflammation of the bile ducts, characterized by epithelial lymphocytes and periductal epithelioid granulomas. There are four stages of PBC.

Stage 1 - Portal venous stage: Normal-sized triads; portal vein inflammation; minimal bile duct damage. Granulomas are usually detected during this stage.

Stage 2 - Periportal Stage: Enlarged triads; periportal fibrosis and/or inflammation. Typically, this stage is characterized by the presence of proliferation of small bile ducts.

Stage 3 - Septal stage: active and/or passive fibrous septa.

Stage 4 - Biliary cirrhosis: Nodules present; Garland

The term "primary sclerosing cholangitis" (PSC) refers to a bile duct disease that causes inflammation and subsequent obstruction of the bile ducts at both the intrahepatic (inside the liver) and extrahepatic (outside the liver) levels. The inflammation impedes the flow of bile to the intestine, which can ultimately lead to cirrhosis of the liver, liver failure, and liver cancer.

The term "non-alcoholic steatohepatitis" (NASH) refers to liver inflammation caused by a buildup of fat in the liver. In some people, the buildup of fat causes liver inflammation. Because of the inflammation, the liver cannot function as well as it should. NASH can become more severe and cause scarring of the liver, which can lead to cirrhosis. NASH is similar to the type of liver disease caused by long-term, heavy drinking. However, NASH can occur in people who do not drink alcohol excessively.

The term "organ" refers to a differentiated structure composed of cells and tissues that performs some specific function of an organism (as in the heart, lungs, kidneys, liver, etc.). The term also encompasses body parts that perform a function or cooperate in an activity (e.g., the eye and related structures that constitute the organ of vision). The term "organ" further encompasses any partial structure of differentiated cells and tissues that can potentially develop into a complete structure (e.g., a lobe or portion of a liver).

All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. The citation of publications and patent documents does not constitute an admission that any of them are relevant prior art, nor does it constitute any admission as to their contents or dates. Having now described the invention by written description, those skilled in the art will recognize that the invention can be practiced in various embodiments, and the description and examples provided herein are for illustrative purposes only and do not limit the appended claims.

In this specification, unless the context clearly dictates otherwise, the singular also includes the plural. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of conflict, the present specification shall prevail. Unless otherwise indicated, all percentages and ratios used herein are by weight.

Examples

Example 1: Bile Duct Ligation (BDL) Model

This experiment was performed to evaluate the effects of OCA and atorvastatin, alone and in combination, on fibrosis induced by common bile duct ligation in mice.

Animals, Confinement, and Diet

Male C57BL/6 mice (6 weeks old) were obtained from SLC, Japan. Animals were maintained in a specific pathogen-free facility under controlled conditions of temperature (23°C ± 2°C), humidity (45% ± 10%), light (12-hour artificial light and dark cycle; light from 8:00 to 20:00) and ventilation (ventilation rate: more than 40 times/hour). High pressure (20 ± 4Pa) was maintained in the laboratory to prevent the equipment from being contaminated. Animals were housed in KN-600 (Natsume Seisakusho, Japan) with a maximum of 6 mice per cage. Sterile clean paper (SLC, Japan) was used for bedding and changed once a week. Sterilized solid high-fat diet (HFD) and water were provided ad libitum 3 weeks before surgery.

Treatment group

Group 1: sham operation

Sham-operated mice (n=8) were orally administered vehicle (0.5% CMC) once daily at a volume of 5 mL/kg from day 0 to day 6 after BDL surgery.

Group 2: BDL-carrier

BDL-operated mice (n=12) were orally administered vehicle (0.5% CMC) once daily at a volume of 5 mL/kg from day 0 to day 6 after BDL surgery.

Group 3: BDL-OCA

BDL-operated mice (n=12) were orally administered vehicle supplemented with OCA once daily at a dose of 5 mg/kg from day 0 to day 6 after BDL surgery.

Group 4: BDL-atorvastatin

BDL-operated mice (n=12) were orally administered vehicle supplemented with atorvastatin at a dose of 10 mg/kg once daily from day 0 to day 6 after BDL surgery.

Group 5: OCA-BDL-atorvastatin

BDL-operated mice (n=12) were orally administered once daily with OCA-supplemented vehicle at a dose of 5 mg/kg and atorvastatin-supplemented vehicle at a dose of 10 mg/kg from day 0 to day 6 after BDL surgery.

Bile duct ligation surgery

At day 0, bile duct ligation surgery was performed. Cholestasis was established in mice by common bile duct ligation under pentobarbital anesthesia, and this cholestasis caused liver fibrosis over time. Based on the weight of mice the day before surgery, mice were divided into two surgical groups. After shaving, the abdominal cavity was opened, and the common bile duct was connected twice with 5-0 surgical silk, and the common bile duct was incised between ligations. The peritoneum and skin were closed with sutures. Mice were transferred to a clean cage (resting cage) for recovery from anesthesia. Sham-operated mice (Sham mice) were operated on in a manner similar to that of other groups, but the bile duct was not ligated.

Animal monitoring and sacrifice

Viability, clinical signs, and behavior were monitored daily. Body weight was recorded daily during the treatment period. Food consumption was measured twice a week per cage during the treatment period. On day 6, animals were sacrificed by exsanguination via direct cardiac puncture under ether anesthesia (Wako Pure Chemical Industries, Ltd.).

Histological analysis

In order to visualize collagen deposition, picro-Sirius red solution (Waldeck, Germany) was used to stain the left liver sections fixed by Bouin's. For quantitative analysis of fibrotic areas, a digital camera (DFC295; Leica, Germany) was used to capture the bright field images of the picro-Sirius red stained sections around the central vein at 100 times magnification, and the positive area of 5 visual fields/sections was measured using ImageJ software (National Institute of Health, USA). Statistical analysis was performed using Prism software 6 (GraphPad Software, Inc., USA).

result

Liver sections were subjected to histopathological analysis (according to conventional methods) by Sirius red staining to estimate the percentage of fibrosis area. Representative micrographs of liver sections stained with Sirius red are shown in Figures 1A-1E. Compared with the BDL-sham group, the BDL-vehicle group showed a significant increase in Sirius red-positive area. As shown in Table 1 and Figure 2, compared with the BDL-vehicle group, the BDL-OCA+atorvastatin group showed a significant decrease in Sirius red-positive area.

Table 1

The results in Table 1 show that the combination of OCA and atorvastatin significantly reduced fibrosis.

Example 2: Diet-induced NASH in APOE*3Leiden.CETP mice

This experiment was conducted to evaluate the effects of OCA and fenofibrate, alone or in combination, on the development of diet-induced NASH and liver fibrosis in APOE*3Leiden.CETP transgenic mice. Liver gene expression profiling and subsequent pathway analysis were performed to determine whether the combination regulates novel genes not regulated by either monotherapy treatment and/or more strongly regulates genes also affected by the monotherapy.

Animals, Confinement, and Diet

Male APOE*3Leiden.CETP transgenic mice (9-21 weeks old) were obtained and housed 2-5 mice per cage. Mice were fed a high-fat diet containing 24% lard and 1% (w/w) cholesterol. The mice were maintained on the high-fat diet for 15 weeks. At week 16, after a 4-hour fast, the mice were paired based on age, body weight, plasma cholesterol, and triglycerides.

Treatment group

Group 1: HFC reference group initial treatment

During the running period from week 0 to week 14, mice (n=15) were fed a high-fat diet.

Group 2: HFC control group

Mice (n=15) were fed a high-fat diet from week 0 to 24.

Group 3: HFC+OCA

From weeks 16 to 24, mice (n=15) were fed a high-fat diet supplemented with OCA at a dose of 10 mg/kg once daily.

Group 4: HFC + low-dose fenofibrate

From weeks 16 to 24, mice (n=15) were fed a high-fat diet supplemented with fenofibrate at a dose of 10 mg/kg once daily.

Group 5: HFC + high-dose fenofibrate

From weeks 16 to 24, mice (n=15) were fed a high-fat diet supplemented with fenofibrate at a dose of 40 mg/kg once daily.

Group 6: HFC+OCA+low-dose fenofibrate

From weeks 16 to 24, mice (n=15) were fed a high-fat diet supplemented with OCA at a dose of 10 mg/kg and fenofibrate at a dose of 10 mg/kg once daily.

Group 7: HFC+OCA+high-dose fenofibrate

From weeks 16 to 24, mice (n=15) were fed a high-fat diet supplemented with OCA at a dose of 10 mg/kg and fenofibrate at a dose of 40 mg/kg once daily.

Group 8: Food control group

Mice (n=8) were fed chow from week 0 to week 24.

Study Design

Mice were fed a high-fat diet (HFC) diet for 14 weeks. After 15 weeks of HFC diet, HFC mice were divided into 7 groups according to age, body weight, plasma cholesterol and triglycerides after fasting for 4 hours. Starting from the 15th week, mice were treated with OCA and fenofibrate alone or in combination and sacrificed at the 25th week in a non-fasted state. One week before sacrifice, mice were labeled with _D2O by intraperitoneal (ip) injection of a large amount of _D2O and then adding 4% _D2O to drinking water. Plasma (EDTA) was obtained by cardiac puncture and stored at -70°C. The liver was weighed and 4 livers were separated: 1 piece (medial lobe) was fixed in 10% formalin (for NASH and fibrosis histology) and 3 pieces (left lobe) were quickly frozen in liquid _N2 and stored separately at -70°C.

Liver inflammation score

Inflammation is a key feature of NASH. Inflammation was classified according to the procedure of Liang et al., PlosOne [Public Library of Science], December 2014, 9 (12) and scored by quantitative analysis of the number of inflammatory cell aggregates. Specifically, the level of inflammation was assessed by counting the number of inflammatory lesions per field of view using 100x magnification (view size 3.1 mm2; average of 5 different fields of view).

Results: Summary of the Effects of OCA+/-Fenofibrate on Inflammation in APOE*3-Leiden.CETP Mice

The effects of OCA 10 mg/kg alone and in combination with fenofibrate (10 and 40 mg/kg) on inflammation in APOE*3-Leiden.CETP mice on a NASH diet were studied. After 10 weeks of drug administration at low doses, neither OCA (10 mg/kg) nor fenofibrate (10 mg/kg) reduced the number of inflammatory cell lesions (Figures 3A and 3B and Table 2). In contrast, the combination significantly reduced inflammation relative to the vehicle control and each monotherapy group. A higher dose of fenofibrate (40 mg/kg/d) also significantly reduced inflammation relative to the vehicle control. No additional anti-inflammatory effect was apparent when combined with OCA, as high-dose fenofibrate produced a near-maximal effect on its own. In summary, a significant reduction in inflammation was observed with the OCA + low-dose fenofibrate combination (-63%). In addition, a significant reduction was observed with high-dose fenofibrate (-74%), and a similar reduction was observed with the combination of OCA (-79%). See Table 2 and Figures 3A and 3B.

Table 2

The results in Table 2 indicate that the efficacy of the combination of OCA and high-dose fenofibrate is driven by and reaches a ceiling with high-dose fenofibrate.

RNA isolation and sequencing

Nucleic acid extraction was performed as previously described in detail (Verschuren et al., 2014). Briefly, total RNA was extracted from individual liver samples using glass beads and RNAzol (Campro Scientific, Veenendaal, the Netherlands). RNA concentration and quality were determined using a fragment analyzer (Advanced Analytical Technologies, USA) and an RNA 6000 Nano Lab-on-a-Chip kit and a Bioanalyzer 2100 (Agilent Technologies, Amstelveen, the Netherlands). All samples met quality requirements and were used for RNA sequencing procedures.

The samples were processed using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina. Sample preparation was performed according to the experimental protocol "NEBNext Ultra Directional RNA Library Prep Kit for Illumina" (NEB#E7420S/L). In brief, mRNA was isolated from total RNA using oligo-dT magnetic beads. After mRNA fragmentation, cDNA synthesis was performed. This was used for connection of sequencing adapters and PCR amplification of the resulting products. The quality and yield of the samples after preparation were measured using a fragment analyzer (Advanced Analytical Technologies, Inc., USA). The size of the resulting products was consistent with the expected size distribution (broad peak between 300-500bp).

Aggregation and DNA sequencing were performed using an Illumina NextSeq 2500 according to the manufacturer's protocol. Data were generated using a single-end read sequencing protocol, resulting in approximately 15 million reads per sample and 75 bp per read. Image analysis, base calling, and quality checks were performed using the Illumina data analysis pipeline RTA v2.4.11 to generate raw data (*.fastq files).

The reads were mapped to the reference sequence Mus musculus GRCm38.p3 using a short read aligner based on the Burrows-Wheeler transformation. A default mismatch rate of 2% (3 mismatches in 150 base reads) was used. Based on the read positions mapped in the alignment file (*.bam-file), the frequency of how often the reads were mapped on the transcripts was determined. The counts were saved to a count file, which served as input for downstream mRNA-seq differential expression analysis. The read counts were loaded into the DESeq software package, which is a statistical software package within the R platform. DESeq was specifically developed for standardizing RNA-seq data from different samples and finding differentially expressed genes between two conditions of RNA-seq data to estimate the relationship between the mean and variance of each gene (Anders et al., 2013). In addition, it allows scaling factors to be easily included in statistical tests. Differentially expressed genes were identified using a significance threshold of P < 0.01 and used as input for pathway analysis using the Ingenuity Pathway Analysis (IPA) suite (accessed 2016).

Upstream regulator analysis is carried out using IPA software (Kramer et al., 2014). The analysis determines the activation state of transcription factors based on the differential gene expression observed. This produces overlapping P values and activation z scores for each transcription factor in the IPA knowledge base. The overlapping P value represents the importance of overlapping between the known target genes of the transcription factor and the differentially expressed genes measured in the experiment. The activation z score represents activation (positive z score) or inhibition (negative z score) of a specific transcription factor. Activation z score <-2 or >2 indicates significant inhibition or activation of a pathway or process.

Omics results

Next generation sequencing of liver mRNA samples from treated mice was performed to gain insight into the underlying mechanisms and pathways.Two analyses were performed to gain insight into the underlying mechanisms and pathways.

First, the enrichment analysis of standard pathway analysis shows that OCA regulates several inflammatory processes (Fig. 6 A). The figure plots each pathway as a function of -log p value (for reference, the conversion value of p<0.05 is 1.3, the conversion value of p<0.0001 is 4, the conversion value of p<0.000005 is 5.3 etc.). The regulatory pathway with OCA monotherapy is related to T cell and B cell signaling, leukocyte extravasation signaling, natural killer cell signaling, etc. Low-dose fenofibrate has no effect on these pathways. When OCA is combined with low-dose fenofibrate, some identical pathways are strongly regulated (in the case of phagocytosis and phagosome formation of FC receptor-mediated in iCOS-iCOSL signaling, leukocyte extravasation signaling, pattern recognition receptors, macrophages in helper T cells). In addition, other pathways (e.g., cholesterol biosynthesis I and II, fatty acid b oxidation) (Fig. 6 B) that are not significantly regulated by any single agent are regulated by the combination. Consistent with the histological data, high-dose fenofibrate had potent effects on these pathways, but these effects were not enhanced by co-administration with OCA.

A more detailed analysis was performed around the pathways involved in leukocyte extravasation signaling. Leukocyte extravasation is essential for the pathophysiological processes in NASH (and other diseases). These processes include the migration of T lymphocytes for immune surveillance, the recruitment of activated lymphocytes and granulocytes during acute and chronic inflammatory responses, and the homing and mobilization of hematopoietic progenitor cells. The effects of maintaining mice on a high-fat diet from this study illustrate these processes that significantly up-regulated and down-regulated genes. Table 3 describes the effects of a high-fat diet on leukocyte extravasation signaling in mice maintained on a high-fat diet relative to mice maintained on a standard chow, as well as the effects of the combination therapy relative to a high-fat diet.

Table 3.

Leukocyte migration and extravasation across the endothelium occur in several distinct steps, including rolling of leukocytes on endothelial cells, mediated by transient, weak interactions between adhesion molecules. Subsequently, loosely attached leukocytes become so close to the endothelium that they are activated by chemotactic cytokines presented on the apical surface of the endothelium. Next, the activated leukocytes spread and firmly attach to docking structures formed by the endothelium, ultimately migrating through the intercellular spaces between endothelial cells into the underlying tissue.

Administration of OCA downregulated many genes involved in the inflammatory cascade in leukocytes (WAP, Rac2, RASGRP1, Vav, PKC, PI3K, ERM, ITGAL, and PSGL-1) and in endothelial cells (VCAM1, PI3K, ERM, NOX, CYBA, PKC, NCF1, and NCF2). Regulation of genes in these pathways was not evident after low-dose fenofibrate alone.

When OCA was combined with fenofibrate at doses that were ineffective in modulating these pathways, multiple additional genes were now regulated, indicating a synergistic effect. In leukocytes, these additional genes included CD43, PSGL-1, CXCR4, ITGAM, ITGB2, Rap1, ITGA4. In endothelial cells, these additional genes included ICAM1, RhoGAP, VASP, NCF4, ITGAM, ITGB2, ITGA4, and ICAM-1.

As described above, high dose fenofibrate had numerous effects on this pathway that were not augmented by combination administration with OCA. All subsequent analyses focused on low dose monotherapy and combination (OCA 10 mg/kg +/- fenofibrate 10 mg/kg).

In the second analysis, the genes that were differentially regulated between the low-dose combination and each respective monotherapy were compared. This is different from the first gene expression analysis (described above) focusing on the comparison relative to the vehicle group, and the following analysis compares each monotherapy with the combination. The Venn diagram (Fig. 7A) shows that OCA has 109 uniquely regulated genes, and fenofibrate has 92 uniquely regulated genes and 6 co-regulated genes. 517 overlapping genes were regulated with the combination of OCA, 75 genes were regulated with the combination of fenofibrate, and 5 genes were all shared. It is noteworthy that the combination regulated a total of 912 unique genes. Subsequent pathway enrichment highlighted the biological processes (Fig. 7B) in which the combined genes participated.

Pathway analysis was then performed for leukocyte extravasation (e.g., as above), but this time the comparison was between the combination and each monotherapy. For leukocyte extravasation, comparison of the combination with each monotherapy revealed a number of genes that were uniquely regulated in combination-treated mice, consistent with the enhanced anti-inflammatory changes observed histologically (Table 4).

Table 4.

Given the progression from inflammation to fibrosis in NASH and the observation that liver fibrosis/HSC pathways appeared significantly regulated in the combination, we also examined pathways in HSCs. When compared to the monotherapy, it was clear that more genes were regulated in the combination compared to fenofibrate alone, and fewer genes were regulated in the combination compared to OCA. In other words, there was a clear interaction between the two agents with respect to these fibrosis pathways, but the OCA portion of the combination was likely driving these effects more strongly.

Explanation and relevance

The importance of FXR activation in preventing fibrosis and inflammation in livers from FXR knockout mice has been demonstrated (Yang 2007), which exhibit elevated expression of inflammatory genes associated with progressive age-related damage and inflammation (Kim 2007). Consistent with these reports, OCA exerts anti-inflammatory properties in HepG2 cells and primary mouse hepatocytes. HepG2 cells pretreated with OCA and then exposed to pro-inflammatory stimuli showed a 50% to 60% reduction in TNF-α mRNA levels, cyclooxygenase-2 (COX-2) induction, and TNF-α-stimulated inducible nitric oxide synthase (iNOS) expression. Similarly, primary hepatocytes treated with OCA showed blunted induction (40% to 50%) of iNOS and monocyte chemoattractant protein-1 (MCP-1) gene expression in response to pro-inflammatory stimuli (Wang 2008). The effects of OCA on the mechanisms of cell migration have not been directly investigated, but OCA inhibited the production of iNOS or COX-2 induced by IL-1β and abolished pharmacologically induced migration of rat aortic smooth muscle cells (Li 2007). Similar inhibition of inflammatory infiltrates by OCA has been demonstrated in intestinal tissue from two animal models of inflammatory bowel disease (DSS and trinitrobenzenesulfonic acid) (Gadaleta 2011) and in renal tissue from a rat model of type 1 diabetes (Wang 2010). Therefore, the observation of enhanced anti-inflammatory effects by OCA suggests that the gene expression changes associated with the combination of OCA and fenofibrate could enhance the inhibition of inflammation and inflammatory cell migration in many disease conditions.

Example 3: Diet-induced NASH in leptin-deficient ob/ob mice

This experiment was performed to evaluate the effects of 8 weeks of treatment with OCA and atorvastatin, alone and in combination, on the stage of fibrosis (pre-biopsy vs. post-biopsy) in male leptin-deficient ob/ob-NASH mice.

Animals, Confinement, and Diet

Male Lep ^ob / Lep ^ob mice (5 weeks old) were purchased from Jan Vier, France. During the acclimation and diet induction period, mice were housed in custom-made cabinets in groups of five per cage under a 12:12 light-dark cycle (with light on from 3 AM to 3 PM) under controlled temperature conditions (22 ° C ± 1 ° C; 50% ± 10% relative humidity). Throughout the diet induction and study period, mice were given a custom NASH diet (S8189, Ssniff, Germany) (40% fat, 40% carbohydrates (20% fructose) and 2% cholesterol) or regular rodent chow (ob / ob-CHOW) (Altromin 1324, Brogaarden, Denmark) and tap water at will. Animals were kept on a diet for a total of 18 weeks before intervention and maintained on a diet throughout the study. During postoperative recovery and throughout the study, animals were housed individually.

Treatment group

Group 1: Lep ^ob / Lep ^ob -NASH carrier

From week 0 to week 8, mice (n=10) were orally administered vehicle (0.5% CMC) once daily at a volume of 5 mL/kg.

Group 2: Lep ^ob /Lep ^ob -NASH OCA

Mice (n=10) were orally administered vehicle supplemented with OCA at a dose of 30 mg/kg once daily from week 0 to week 8.

Group 3: Lep ^ob / Lep ^ob -NASH

Mice (n=11) were orally dosed with vehicle supplemented with atorvastatin at a dose of 10 mg/kg once daily from week 0 to week 8.

Group 4: Lep ^ob / Lep ^ob -NASH OCA + atorvastatin

Mice (n=9) were orally administered once daily a combination of vehicle supplemented with OCA at a dose of 30 mg/kg and atorvastatin supplemented with a dose of 10 mg/kg.

Allocation to the study, stratified randomization, and baseline monitoring

After 15 weeks of dietary induction (3 weeks before the start of the study), liver biopsies were obtained to assess the liver progression of fibrosis and steatosis and for liver fibrosis stage assessment. At week 1, patients were randomly assigned to treatment groups stratified by liver fibrosis stage, steatosis score, and body weight.

Pre-biopsy procedure

On the day of operation, mice were anesthetized with isoflurane (2%-3%) in 100% oxygen. A small laparotomy was performed in the midline and the left lateral lobe of the liver was exposed. A tapered wedge of liver tissue (approximately 100 mg) was excised from the distal portion of the lobe, weighed, and fixed in 4% paraformaldehyde (PFA) for histology. The cut surface of the liver was immediately electrocoagulated using bipolar coagulation (ERBE VIO 100 electrosurgical scalpel). The liver was returned to the abdominal cavity and the abdomen was sutured and the skin was closed with a suturer. On the day of operation, mice were given warm saline (0.5 ml) for rehydration. For postoperative recovery, carprofen (5 mg/ml-0.01 ml/10 g) and enrofloxacin (5 mg/ml-1 ml/kg) were given subcutaneously on the day of operation and on the 1st and 2nd day after surgery.

Pre-screening to assess liver levels of steatosis and fibrosis

Liver biopsy slice preparation for histological assessment: after overnight storage in 4% PFA, liver biopsy slices are infiltrated in the paraffin in the automatic Miles Scientific Tissue-TEK VIP-TEK tissue processor overnight and are subsequently embedded in paraffin blocks. Biopsy slices from five different animals are embedded on a block. Then, these blocks are trimmed and two 3 μm sections (one for Sirius red staining, and one for H & E staining) are cut on a Microm HM340E ultramicrotome (Thermo Scientific). Two blocks are placed on a slide, thereby obtaining 10 biopsy slices totaling to represent 10 different animals for each slide. Sections are left to dry overnight. As outlined by the people such as Kleiner (2005) (vide infra), (vide infra) stratified and randomized to the evaluation of the fibrosis stage of the treatment group.

Baseline and final plasma biomarkers

Blood samples for measuring non-fasted (fed) plasma levels of triglycerides were obtained in the morning (7-8 AM) at baseline and treatment week 8. Blood samples were collected from the tail vein (by shearing) in a conscious state. The most recent drug dose was given approximately 18 hours before blood sampling. Mice were fed again after blood sampling.

Termination and autopsy

Animals were terminated in a non-fasting state at week 8. The most recent drug dose was given approximately 18 hours prior to termination and animals were not administered drugs prior to termination. Animals were induced with CO ₂ /O ₂ and during anesthesia (isoflurane), the abdominal cavity was opened and cardiac blood was obtained for collection of terminal plasma. After necropsy, whole livers were collected and weighed. Biopsies were removed from the left lateral lobe and fixed in 4% PFA for histological and biochemical analysis. The median lobe was divided into slices and rapidly frozen in liquid nitrogen for biochemical analysis (TG). The remaining liver tissue was subsequently fixed in 4% PFA for later optional histology.

Liver tissue processing

Pre-study biopsies: Approximately three weeks prior to study initiation, tapered wedges of liver tissue (approximately 100 mg) were resected from the distal portion of the left lateral lobe, weighed, and immediately placed in 4% PFA.

Terminal liver tissue: After 8 weeks of treatment, whole livers were collected, weighed, and liver biopsies were removed from the left lateral lobe and immediately placed in 4% PFA (approximately 150-200 mg). Several slices of the mid-lobe were quickly frozen in cryovials (RNAseq) (approximately 100 mg) and FastPrep tubes and used for TG (approximately 100 mg) and TC (approximately 50 mg).

For histological fixation, embedding and sectioning: after overnight fixation in 4% PFA, liver biopsy sections were immersed in paraffin in the automatic Miles Scientific Tissue-TEK VIP-TEK tissue processor overnight and embedded in paraffin blocks subsequently. Biopsy sections from five different animals were embedded in a block. These blocks were trimmed and two 3 μm sections/blocks were cut on a Microm HM340E ultramicrotome (Thermo Scientific). A section from two different blocks was placed on an object slide, providing a total of 10 biopsy sections per slide as outlined above.

Fibrosis stage

Liver pre- and post-biopsy tissues from the left lateral lobe were collected for assessment of fibrosis stage using the clinical criteria outlined by Kleiner and colleagues (Design and validation of a histological scoring system for nonalcoholic fatty liver disease, Kleiner et al., Hepatology 41; 2005) and are reproduced in Table 5 below. Figure 4 depicts the effects of OCA and atorvastatin alone and in combination on fibrosis scores. The combination of OCA and atorvastatin showed a trend toward lowering fibrosis scores, although not in a significant manner from vehicle (p value = 0.09).

Table 5

Plasma triglycerides

Triglyceride levels: 100 μl of blood was collected into lithium-heparin tubes. Plasma was separated and samples were stored at -80 degrees Celsius until analysis. Triglyceride levels were measured in a single assay using an automated analyzer Cobas C-111 and a commercial kit (Roche Diagnostics, Germany) according to the manufacturer's instructions. As shown in Figures 5A and 5B, the combination of OCA and atorvastatin reduced triglyceride levels in a statistically significant manner.

Example 4: Sandwich culture of hepatocytes

This experiment will be performed to evaluate the effects of OCA in combination with PPAR agonists or statins to determine their ability to alter collagen synthesis in human hepatocytes.

Reagents and solutions

The cell culture medium that is applicable to comprises is Eagle's culture medium, Williams' culture medium E, Leibovitz's culture medium L15 and the Xu's culture medium of improvement of Wei Mosishi (Waymouth's), Ham's (Ham's) F12, RPMI 1640, Duchenne's improvement.IV collagenase, type I collagen, Percocet, culture medium and supplement can be added into culture medium (for example, serum, antibiotic, amino acid, hormone such as DEX, insulin and somatomedin), and perfusion buffer and other solutions are commercially available or are made up of commercially available materials.Other types of collagen (II-IV type), laminin, fibronectin and heparin sulfate proteoglycan can be used for sandwich hepatocyte culture.But, shown that type I and type IV collagen are better than fibronectin and laminin.

Isolation of hepatocytes

A two-step in situ collagenase perfusion method will be used to isolate hepatocytes. Briefly, hepatocytes will be isolated from female Lewis rats. The animals will be anesthetized. The liver will first be perfused in situ with perfusion buffer via the portal vein. The perfusate will be equilibrated before entering the liver. The liver will then be perfused with collagenase in perfusion buffer. The liver will then be dissected and transferred to ice-cold perfusion buffer. The liver capsule will be opened and the resulting cell suspension will be filtered. The cell pellet will be collected by centrifugation and resuspended. Percol will be added to the suspension, and the hepatocytes will be isolated using a Percol density centrifugation technique. The mixture will be centrifuged, and the cell pellet will be washed twice with culture medium. Hepatocyte viability will be determined by trypan blue exclusion. Alternatively, cryopreserved hepatocytes can be used instead of freshly isolated hepatocytes.

Sandwich culture of hepatocytes

The isolated hepatocytes were cultured on collagen-coated tissue culture plates and maintained in a culture medium supplemented with serum, penicillin, streptomycin, epidermal growth factor, insulin, glucagon, and hydrocortisone. A collagen gelling solution was prepared by mixing a type I collagen solution with the culture medium. The tissue culture plates were coated with the gelling solution and incubated at 37°C to promote gel formation. The hepatocytes were seeded at an appropriate density and maintained at 37°C. The culture medium was replaced every 24 hours.

For the sandwich system, an additional collagen gel solution was distributed over the cells after one day of culture. The culture medium was carefully removed to ensure that the second layer of collagen gel was evenly distributed across the plate. The plates were incubated at 37°C to allow for gelation and adhesion of the second gel layer before the culture medium was changed. The culture medium was changed daily. Culture medium samples were stored at -20°C for further analysis.

Hepatocytes cultured between multiple layers of gel collagen maintained a three-dimensional cubic shape and a distribution of cytoskeletal proteins similar to that observed in vivo.

Optimization of bile canalicular network formation

To optimize taurocholate accumulation and bile excretion, specialized media such as Williams' medium E and Dulbecco's modified Eagle's medium can be used for intercalated hepatocyte culture.

Test samples

The FXR agonists targeted for study were obeticholic acid, also known as "OCA," and 6-ethylchenodeoxycholic acid (6-ECDCA).

The PPAR-α agonists intended for investigation include one or more of clofibrate, gemfibrozil, ciprofibrate, bezafibrate, and fenofibrate.

The dual PPAR-α/δ agonist is 2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1(E)-propenyl]phenoxy]-2-methylpropanoic acid.

The PPAR delta (δ) agonist intended for use in the study was GW501516.

Statins (HMG-CoA reductase inhibitors) intended for investigational use include atorvastatin (Lipitor), rosuvastatin (Crestor), and simvastatin (Zocor).

Example 5: Evaluation of the effect of single administration of a test sample on lipid profile

The potential of 5 test samples, namely FXR agonists, PPAR-α agonists, PPAR-δ agonists, dual PPAR-α/δ agonists (or alternatively, PPAR-α agonists and PPAR-δ agonists together) and statins will be evaluated to determine the ability to alter cholesterol synthesis and lipid profiles in human hepatocytes. Changes will be assessed in sandwich cultured human hepatocytes (SCHH) after 72 hours of exposure to the test samples at 3 different concentrations. Dosing solutions will be freshly prepared in culture medium each day, and dosing of SCHH will occur daily for 3 days. One (1) batch of Transporter Certified ^™ human hepatocytes (N=1) will be used to perform the experiment in a 24-well format. Each test condition will be performed in three (3) wells to provide triplicate data (expressed as mean ± standard deviation). Solvent control treated plates will be used as controls and to assess baseline function. At the end of the test period, internal standards will be added to each well, followed by extraction reagents for comprehensive lipid profile analysis. The samples will be shaken at room temperature for 1 hour and centrifuged. The supernatant was evaporated to dryness under nitrogen, resuspended and analyzed.

Comprehensive lipid profile analysis is performed using ultra-high performance liquid chromatography (UPLC) and high-resolution MS. The methyl tert-butyl sample extract will be analyzed on a UPLC-MS (Synapt G2Ion-Mobility QToF) instrument in ESI+ and ESI- modes to cover a wide range of lipid polarity and chemical composition. Initially, UPLC will be used to evaluate the complex effects of large arrays (5000 to 8000) of lipids (including a variety of cholesterol esters). Colorimetric analysis will be performed to measure total cholesterol. Based on abundance, most will be glycerophospholipids, however, a large number of species will be evaluated. Features will be evaluated to identify the potential effects on individual lipids. Standards can be identified using retention time, accurate mass, and fragmentation to identify specific lipids. According to the results, specific lipids or lipids can be identified for the evaluation in Examples 6 and 7. Confirmatory studies will be repeated in two batches of other Transporter Certified ^™ human hepatocytes (N=2).

Example 6: Evaluation of the Effect of Dual Combinations of Test Samples on Lipid Profiles

The potential of FXR agonists in combination with each of a PPAR-α agonist, a PPAR-δ agonist, a PPAR-α and δ dual agonist (or alternatively, an FXR agonist with a PPAR α agonist and a PPAR δ agonist) and/or a statin to alter cholesterol synthesis and lipid profiles in human hepatocytes will be evaluated. Specific combinations evaluated will be:

FXR agonists and PPAR-α agonists

FXR agonists and PPAR-δ agonists

FXR agonists with dual PPAR-α and δ agonists, and/or FXR agonists with both PPAR-α and PPAR-δ agonists

FXR agonists and statins

Changes will be assessed in sandwich cultured human hepatocytes (SCHH) after 72 hours of exposure to the test samples at 3 different concentrations. Dosing solutions will be prepared fresh in the culture medium each day, and dosing of SCHH will occur daily for 3 days. The experiment will be performed in a 24-well format using one (1) batch of Transporter Certified ^™ human hepatocytes (N=1). Each test condition will be performed in three (3) wells to provide triplicate data (expressed as mean ± standard deviation). Samples will be prepared and analyzed for comprehensive lipid profiles as detailed in Example 5. Changes in lipid profiles and cholesterol synthesis will be compared to the effects of administration alone in Example 2.

Example 7: Evaluation of the Effect of Triple Combinations of Test Samples on Lipid Profiles

The potential of a triple combination of FXR agonists, PPAR-α agonists, PPAR-δ agonists, PPAR-α and δ dual agonists (or a combination of a PPAR-α agonist and a PPAR-δ agonist) and/or statins to alter cholesterol synthesis and lipid profiles in human hepatocytes will be assessed. Changes will be assessed in sandwich cultured human hepatocytes (SCHH) after 72 hours of exposure to the test samples at 3 different concentrations. Dosing solutions will be freshly prepared in culture medium daily, and dosing of SCHH will occur daily for 3 days. One (1) batch of Transporter Certified ^™ human hepatocytes (N=1) will be used to perform the experiment in a 24-well format. Each test condition will be performed in three (3) wells to provide triplicate data (expressed as mean ± standard deviation). Samples will be prepared and the overall lipid profile will be analyzed as detailed in Example 2. The changes in lipid profile and cholesterol synthesis will be compared to the effects of the combination given in Example 2. The specific combinations evaluated will be:

FXR agonists vs. PPAR-α agonists and statins

FXR agonists vs. PPAR-δ agonists and statins

FXR agonists with dual PPAR-α and δ agonists (or, alternatively, PPAR-α agonists and PPAR-δ agonists) and statins

Samples will be prepared and analyzed for global lipid profiles as detailed in Example 4. Changes in lipid profiles and cholesterol synthesis will be compared to the effects of the combination administered in Examples 4 and 5.

Example 8: Animal Studies

animal

Animals are housed separately in standard cages at 22 ℃ in 12:12-hour light-dark cycles. Male C57BL/6 mice (Jackson Laboratories, Bar Harbor, Maine) are allowed to arbitrarily obtain a diet (Research Diets, New Brunswick, New Jersey, catalog number (Cat. No.) D09100301) rich in fat (40%kcal, Primex partially hydrogenated vegetable oil shortening), fructose (22% by wt) and cholesterol (2% by wt). A low-fat diet (10%kcal, hereinafter referred to as LFD) without fructose or cholesterol will be used as a control diet (Research Diets, Inc., catalog number (Cat. No.) D09100304). Use this verified LFD to set up a group of control mice, which maintains a "normal" liver phenotype for comparison with the animal of the experimental diet.

Treatment group

Control HFD:

Comparison with LFD:

HFD + FXR agonist:

HFD + PPARα (ie, fenofibrate, gemfibrozil, bezafibrate, or ciprofibrate):

HFD + PPARδ (i.e., GW501516):

HFD+PPARα+PPARδ:

HFD + dual PPARα/δ (i.e., GFT505):

HFD plus statin (ie, atorvastatin, simvastatin, rosuvastatin)

HFD+FXR agonist+PPARα:

HFD+FXR agonist+PPARδ:

HFD+FXR agonist+PPARα+PPARδ:

HFD+FXR agonist+dual PPARα/δ:

HFD+FXR agonist+statin:

Histological and digital imaging analysis

At termination, the right middle lobe and/or left lateral lobe of the liver (≥50% of each lobe harvested) will be excised and fixed in 10% neutral buffered formalin (at least 7 days at room temperature). Liver tissue will be carefully excised to select similar sized sections representing both the edge and center of the tissue. Liver tissue will be paraffin embedded, sliced (5 μm) and mounted. Hematoxylin and eosin staining will be used for morphological analysis, and Masson’s trichrome and Sirius red staining will be used to assess liver fibrosis. Histopathological analysis will be performed by a pathologist who is unaware of the study. NAFLD and NASH will be scored using the criteria outlined by Kleiner and colleagues. For quantitative assessment of fibrosis, the entire Sirius red-stained section will be scanned at x20 magnification using a ScanScope CS whole slide scanning system (Aperio, Vista, California). Image will be extracted and by using Image-Pro analyzer software (MediaCybernetics v.6.2, Bethesda, Maryland) to measure the collagen profile of Sirius red staining from whole tissue based on the color method of cube.Total collagen staining (reported as % of total area) from three to four representative sections of each animal will be assessed (except comprehensive liver fibrosis assessment experiment, wherein other section will be assessed).All histological analyses will be carried out in a blinded manner.

Liver biopsy

Mice will be anesthetized with isoflurane (2%-3%) in 100% oxygen. A small laparotomy will be performed approximately 0.5 cm to the left of the midline, and the left lobe of the liver will be exposed. A wedge-shaped liver tissue (approximately 50 mg) will be excised from the distal portion of the lobe, immediately placed in a vial, and quickly frozen in liquid nitrogen. A wedge-shaped absorbent gelatin sponge (GelFoam, Pfizer, New York) will be inserted into the cut edge of the liver. Once hemostasis is achieved (usually within 1 minute) and the gelatin sponge adheres well to the biopsy site, the liver will be returned to the abdominal cavity, the abdominal wall will be sutured, and the skin will be stapled. During surgery, mice will receive a single injection of buprenorphine (0.05 mg/kg, subcutaneous) to control postoperative pain. Sham-operated mice will undergo the same process, except that no incision is made in the liver.

Plasma and serum analysis

Plasma glucose, triglycerides, total cholesterol, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels will be measured by using an Olympus AU400e bioanalyzer (Olympus America Diagnostics, Center Valley, PA). Plasma samples will be diluted 1:10 with PBS for measurement of ALT and AST. Total plasma adiponectin and fasting serum insulin will be measured using commercially available electrochemiluminescence kits (Meso Scale Discovery, Gaithersburg, MD) according to the manufacturer's instructions.

Quantification of total hepatic lipid and collagen content

Total hepatic lipids will be extracted from the liver using a protocol adapted from Folch et al. Frozen liver tissue (approximately 0.3 g) will be homogenized in 10 ml of a 2:1 chloroform-methanol solution. The homogenate will be filtered using fat-free filter paper and concentrated into a pre-weighed 15-ml glass vial. An additional 5 ml of 2:1 chloroform-methanol will be added, followed by 2.5 ml of 0.9% NaCl. Lipids will be separated by centrifugation at 1,800 g, 10°C for 5 minutes, the aqueous layer discarded, and the tubes flushed with nitrogen until the lipid pellet is dry. The tubes containing the lipid pellet will be reweighed, and the total lipid extracted per gram of total liver will be calculated. Total collagen content in the liver will be measured by acid hydrolysis of collagen (Quickzym, Leiden, the Netherlands) and colorimetric determination of hydroxyproline residues.

Determination of extractable collagen-1α1 protein by Western blotting

Tissue cores (50-100 mg) will be collected from the left lateral lobe of the liver, quickly frozen in liquid nitrogen, and stored at -80 ° C until processed. The tissue will be homogenized in a lysis buffer containing protease inhibitors. The protein concentration of the clarified supernatant will be measured using a BCA protein assay kit (Pierce, Rockford, Illinois). Liver tissue lysates (approximately 50 μg) will be separated and transferred to a nitrocellulose membrane on a reduced 4%-12% Nupage gel (Life Technologies, Carlsbad, California). The membrane will be cut between the 50-kDa and 60-kDa markers and blocked with 5% Blotto. The upper part will be probed with anti-collagen-1%1 (1:1,000; catalog number NBP1-30054; Novus Biologicals, Littleton, Colorado), which detects the COOH-terminal telopeptide portion of the collagen-1%1 protein. For normalization, the lower part will be probed with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:7,500; catalog number 3683; Cell Signaling Technology, Danvers, Massachusetts). After incubation with horseradish peroxidase anti-rabbit antibody, protein expression will be detected using enhanced chemiluminescence (ThermoScientific, Rockford, IL) and optical density will be determined using the FluorChem system (Cell Biosciences, Santa Clara, CA). The optical density of collagen-1α1 will include a 140-kDa mature protein and a slightly larger band corresponding to glycosylated forms or partially processed collagen-1α1 protein.

Changes in hepatic gene expression

Tissue samples from the left lateral lobe of the liver were harvested using a 6-mm tissue coring tool or by biopsy, quickly frozen in liquid nitrogen, and stored at -80°C until processed. Total RNA (approximately 50-150 mg) from liver samples will be extracted using TRI reagent (Life Technologies), and then further purified using Qiagen RNeasy Plus Mini kit (Qiagen, Valencia, California). RNA integrity will be determined on a bioanalyzer 2100 (Agilent Technologies, Santa Clara, California) by using an Agilent 6000 nano kit. cDNA will be prepared using a high-capacity cDNA reverse transcription kit (Life Technologies). Changes in gene expression will be confirmed by using the on-demand TaqMan gene expression assay and universal premix (Life Technologies) on an ABI Prism 7900HT instrument (Applied Biosystems, Foster City, California). The variation of gene expression will be calculated by the comparative threshold cycle (CT) method of peptidyl prolyl isomerase A (Ppia) and Gapdh used for standardization.For gene array, by using ABI Prism 7900HT fast real-time PCR system (Applied Biosystems), cDNA sample is run on mouse fibrosis RT2 analyzer PCR array (PAMM-120C, RT2SYBR Green/ROX qPCR premix; SABiosciences company).The variation of gene expression on the array will be calculated by the comparative CT method of using DataAssist v3.0 software (Applied Biosystems/Life Technologies).In the five housekeeping genes included in mouse fibrosis RT2 analyzer PCR array, according to the stability score calculated by DataAssist v3.0 software, hypoxanthine phosphoribosyl transferase 1 (Hprt) and Gapdh have the most stable expression.The mean value of the endogenous control gene selected is used as normalization factor to calculate the relative expression of each gene. To confirm the results obtained using the fibrosis array, TaqMan gene expression assays will be performed to select genes identified by the array as being upregulated, downregulated, or unchanged.

Example 9: Clinical Trial

Methods: A multicenter, double-blind, placebo-controlled, parallel-group, randomized clinical trial was conducted in patients with noncirrhotic, nonalcoholic steatohepatitis to evaluate oral obeticholic acid (25 mg daily) or placebo for 72 weeks. Patients were randomly assigned in a 1:1 ratio using a computer-generated centrally administered algorithm, stratified by clinical center and diabetes status. The primary outcome measure was improvement in centrally scored liver histology, defined as a reduction of at least 2 points in the nonalcoholic fatty liver disease activity score without worsening fibrosis from baseline to the end of treatment. The change in alanine aminotransferase was measured at 24 weeks: the relative change in alanine aminotransferase was -24%, with a 95% CI of -45 to -3.

Study Design and Participants

Patients were enrolled in the study according to the following inclusion criteria: 18 years of age or older at screening, histological evidence of definite or borderline nonalcoholic steatohepatitis based on a liver biopsy obtained 90 days or less before randomization, and a histological nonalcoholic fatty liver disease (NAFLD) activity score of 4 or greater, with a score of 1 or greater in each component of the score (steatosis score of 0-3, bulging score of 0-2, and lobular inflammation score of 0-3). Biopsy grading and staging for enrollment purposes were performed at the participating site. Exclusion criteria included the presence of cirrhosis, other causes of liver disease, heavy alcohol consumption (>20 g/day for women or >30 g/day for men), or other confounding conditions (see below).

Randomization and masking

Patients who met the eligibility criteria were randomly assigned (1:1) to oral obeticholic acid (25 mg once daily) or placebo. Obeticholic acid and placebo were provided as identical tablets in identical containers labeled with a code. Patients, investigators, clinical site staff, and pathologists were masked to treatment assignment.

process

After randomization, the patient returned for a study visit at the 2nd, 4th and 12th week, then returned every 12 weeks until the 72nd week treatment was completed, and then returned every 24 weeks thereafter. In these visits, blood samples were obtained to be used for routine biochemical tests and to assess the fasting concentrations of lipids, glucose and insulin. Body weight, height and waist and hip circumference were measured at the temporary time of initial assessment and appointment. All patients received standard advice on healthy eating habits, weight loss, exercise and the management of hypertension, hypercholesterolemia and diabetes.

Baseline and end-of-treatment liver biopsies were centrally assessed for consensus scores on each component of the NAFLD activity score, had fibrosis stage determined, and were assigned a diagnosis of NAFLD, borderline NAFLD, or not NAFLD.

Inclusion and exclusion criteria

Patients who met any of the following exclusion criteria were considered ineligible: 1) current or history of heavy alcohol consumption for more than 3 consecutive months within 1 year before screening (heavy alcohol consumption was defined as an average of 20 g/day or more for women and 30 g/day or more for men); 2) alcohol consumption that could not be reliably quantified at the site investigator's discretion; 3) use of medications historically associated with NAFLD for more than 2 weeks within 1 year before randomization; 4) previous or planned bariatric surgery or procedures; 5) uncontrolled diabetes defined as HbA1c of 80.3 mmol/mol or higher within 60 days before participation; 6) presence of cirrhosis on liver biopsy; 7) platelet count <100 × 109/L; 8) clinical evidence of hepatic decompensation as defined by the presence of any of the following abnormalities: serum albumin less than 32 g/L, INR greater than 1.3, direct bilirubin greater than 22 2 μmol/L, or a history of esophageal varices, ascites, or hepatic encephalopathy; 9) evidence of other forms of chronic liver disease: hepatitis B as defined by the presence of hepatitis B surface antigen (HBsAg), hepatitis C as defined by the presence of hepatitis C virus (HCV) RNA or positive hepatitis C antibodies (anti-HCV), evidence of progressive autoimmune liver disease as defined by compatible liver histology, primary biliary cirrhosis as defined by the presence of at least two criteria (biochemical evidence of cholestasis based primarily on elevated alkaline phosphatase, the presence of antimitochondrial antibodies [AMA], and histological evidence of non-suppurative destructive cholangitis and interlobular bile duct destruction), primary sclerosing cholangitis, Wilson's disease as defined by ceruloplasmin below the limits of normal and compatible liver histology. disease), alpha-1-antitrypsin (A1AT) deficiency as defined by diagnostic features on liver histology (confirmed by alpha-1 antitrypsin levels below normal, excluded at the discretion of the site investigator), history of hemochromatosis or iron overload as defined by the presence of 3+ or 4+ stainable iron on liver biopsy, drug-induced liver disease as defined based on typical exposures and medical history, known bile duct obstruction, suspected or confirmed hepatocellular carcinoma, or any other type of liver disease other than NASH; 10) serum alanine aminotransferase (ALT) greater than 300 U/L; 11) serum creatinine of 176.8 μm l/L or more; 12) liver biopsy cannot be obtained safely; 13) history of biliary metastases; 14) known positive human immunodeficiency virus (HIV) infection; 15) active, serious medical illness with a life expectancy of less than 5 years; 16) active substance abuse, including inhaled or injected drugs, within the year before screening; 17) pregnancy, planning pregnancy, possible pregnancy and unwilling to use effective birth control during the trial, or breastfeeding; 18) participation in an IND trial within 30 days before randomization; 19) any other condition that, in the opinion of the site investigator, would hinder compliance or prevent completion of the study; or 20) failure to give informed consent.

Statistical analysis

The primary and binary secondary outcomes were analyzed using the Mantel-Haenszel test; continuous secondary outcomes were analyzed using an analysis of variance (ANCOVA) model that correlated the change in continuous outcomes from baseline to 72 weeks with treatment group and with the baseline value of the outcome. Statistical analyses were performed using SAS (SAS Institute 2011, Basic SAS 9.3 Procedure Guide) and Stata (StataCorp 2013, Stata Statistical Software: Release 13).

result

The primary outcome measure was improvement in centrally scored liver histology, defined as a decrease of at least 2 points in the NAFLD activity score without worsening of fibrosis from baseline to the end of treatment. Worsening of fibrosis was defined as any increase in the value within the stage. Secondary histological outcomes included resolution of nonalcoholic steatohepatitis, change in the NAFLD activity score, and changes in the individual scores for hepatocellular bulging, steatosis, lobular and portal inflammation, and fibrosis. Improvement in fibrosis was defined as any decrease in the value within the stage. For the purposes of this analysis, fibrosis stages 1a, 1b, and 1c were considered stage 1. Other secondary outcomes included changes in serum aminotransferase and gamma-glutamyl transpeptidase concentrations from baseline to 72 weeks, the fasting homeostasis model assessing insulin resistance (HOMA-IR), anthropometric measurements (weight, body mass index, waist-to-hip ratio, waist circumference), and health-related quality of life scores.

Example 10: Data Analysis

Subanalyses of the data obtained in Example 9 were performed to assess the effect of statins on low-density lipoprotein cholesterol (LDL-C) levels. The purpose of these secondary analyses was to determine the effect of OCA versus placebo in a subgroup of patients with more severe NASH and to assess the effect of concomitant statin use on serum LDL cholesterol. Subject data were evaluated in three groups as follows:

Group A (n=64, no statin) included subjects in the obeticholic acid (OCA)-treated group who were not taking a statin at baseline (Day 0) and throughout the study up to and including Week 72.

• Group B (n=47, baseline statin) included subjects in the OCA-treated group who were taking statins at baseline and continued to take statins during the study up to and including Week 72.

- Group C (n=23, neostatin) includes OCA-treated subjects who were not taking a statin at baseline but started statin treatment at a time after baseline up to and including Week 72. The following calculations were performed for OCA-treated subjects in Groups A, B, and C.

The mean and median characteristics listed below will be assessed at baseline and Week 72.

o Laboratory values: LDL-C, high-density lipoprotein cholesterol (HDL-C), alanine and aspartate aminotransferases, gamma-glutamyltransferase

Age

oGender: percentage of males, percentage of females

o Percentage of diabetes mellitus

o Histology: fatty degeneration, swelling, inflammation, fibrosis

The mean and median percentage changes from baseline in the above characteristics will be assessed at Week 72.

During OCA treatment, LDL cholesterol increased in patients taking statins at baseline, but levels did not exceed those in placebo-treated patients not taking statins. During OCA treatment, statin administration reversed LDL levels to below pre-OCA baseline levels. As shown in Figure 8, the OCA-associated increase in LDL appeared to be reversed by initiation of statin therapy during OCA treatment.

Claims (5)

1. A pharmaceutical composition comprising... A two-way combination of FXR agonists and bezafibrate; and Optionally, one or more pharmaceutically acceptable carriers. The FXR agonist described therein is a compound having formula (1): and The pharmaceutical composition described herein does not contain any therapeutic agents other than the FXR agonist and bezafibrate. 2. Use of the pharmaceutical composition of claim 1 in the manufacture of a medicament for treating or preventing FXR-mediated diseases or conditions, wherein the FXR-mediated diseases or conditions are selected from primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), portal hypertension, bile acid diarrhea, chronic liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatitis C infection, alcoholic liver disease, liver damage due to progressive fibrosis, liver fibrosis, cardiovascular disease, and hyperlipidemia. 3. Use of the pharmaceutical composition of claim 1 in the manufacture of a medicament for treating or preventing FXR-mediated diseases or conditions, wherein the FXR-mediated diseases or conditions are selected from: resistant primary biliary cirrhosis; primary biliary cirrhosis with associated elevated liver function tests and hyperlipidemia; primary sclerosing cholangitis; non-alcoholic steatohepatitis; chronic liver disease associated with hepatitis B, hepatitis C, or alcohol; hyperlipidemia, wherein the hyperlipidemia is primary hyperlipidemia with or without a genetic component; and hyperlipidemia associated with coronary artery disease, cerebrovascular disease, peripheral vascular disease, aortic aneurysm, or carotid atherosclerosis. 4. Use of the pharmaceutical composition of claim 1 in the manufacture of a medicament for inhibiting or reversing fibrosis. 5. The use as claimed in claim 4, wherein the fibrosis is selected from liver fibrosis, kidney fibrosis, and intestinal fibrosis.