HK1256960A1

HK1256960A1 - Treatment of mixed dyslipidemia

Info

Publication number: HK1256960A1
Application number: HK18116099.6A
Authority: HK
Inventors: C‧L‧比斯盖尔; D‧C‧奥尼
Original assignee: 耀石治疗公司
Priority date: 2015-11-06
Filing date: 2016-11-07
Publication date: 2019-10-04

Description

Treating mixed dyslipidemia

Cross Reference to Related Applications

This application claims priority to U.S. provisional application serial No. 62/252,195 filed on day 6 of 11/2015 and U.S. provisional application serial No. 62/252,147 filed on day 6 of 11/2015, the disclosures of both of which are incorporated herein by reference in their entireties.

Technical Field

The present invention relates to the use of statins in combination with gemcabene for the treatment of mixed dyslipidemia.

Background

The Fredrickson hyperlipidemia classification system (Fredrickson classification system for hyperlipidemia) uses plasma appearance, total cholesterol, and triglyceride values to characterize subjects with one of five types of hyperlipidemia. These five types are I, II, III, IV and V. Type II is further subdivided into type IIa and type IIb, both of which have elevated total cholesterol and LDL-C, and type IIb also has elevated triglycerides.

It has been reported that the prevalence of type IIb hyperlipidemia (type IIb) in the population is estimated to be about 10%. Type IIb is characterized by elevated levels of LDL-C, triglycerides and apolipoprotein B, as well as very low density lipoprotein cholesterol (VLDL-C), medium density lipoprotein cholesterol (IDL) and small, dense LDLs.

Type IIb hyperlipidemia includes acquired combined hyperlipidemia and Familial Combined Hyperlipidemia (FCHL). FCHL is a genetic disease that occurs in approximately 0.3-2% of the population, but an estimated population incidence of up to 5.7% has been reported. Individuals with type IIb hyperlipidemia have an increased incidence of cardiovascular disease, and those with FCHL have a higher incidence of early-onset coronary heart disease. In addition, patients with type IIb have a higher incidence of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH), a form of fatty liver, than patients with type IIa, which occurs due to overproduction and accumulation of hepatic triglycerides. NAFLD, NASH or fatty liver can lead to metabolic complications including elevated liver enzymes, liver fibrosis, cirrhosis, hepatocellular carcinoma and liver failure. Liver failure is life threatening, and there is an urgent need to develop therapies to delay the onset of fatty liver, prevent fatty liver formation, or reverse fatty liver disorders.

Current treatment options for type IIb hyperlipidemia are limited. While statins are very effective in lowering LDL-C, they are generally not as effective in lowering triglyceride levels. Some statins at high dose levels, such as 80mg atorvastatin, significantly reduce triglyceride levels. However, high dose statin therapy can cause muscle pain (myalgia) and patients are often not well tolerated. In addition, high dose statin therapy increases the risk of severe muscle toxicity, such as rhabdomyolysis.

In addition, the use of high dose statins may be contraindicated for certain drugs because certain statins are metabolized by cytochrome P450 enzymes that also mediate the metabolism of other drugs. It is disclosed in the present application that the combination of gemcabene with low to medium doses of statins, leading to a surprising synergistic effect in lowering Triglycerides (TG), may allow the use of lower doses of statins and thus have better safety.

Treatment of type IIb hyperlipidemia has focused on lowering LDL-C levels and triglyceride levels. Treatment typically involves administration of a combination of a cholesterol lowering agent (e.g., a statin) and a triglyceride lowering agent (e.g., a fibrate, niacin, or fish oil). However, commonly used triglyceride lowering agents may be inconvenient or poorly tolerated, for example, fibrates are associated with increased risk of myalgia and muscle toxicity, fish oils require multiple intakes per day, and niacin leads to flushing, especially when administered in combination with statins. As with certain statins, certain fibrates use or activate the cytochrome P4503a4 isoform as part of their catabolic processes, and administration of these drugs in combination may increase the risk of myalgia and muscle injury. A physician may avoid combining statins with fibrates, because of the concern that such a combination may result in a higher risk of muscle damage.

Non-alcoholic fatty liver disease (NAFLD) is becoming more and more prevalent around the world, particularly in western countries. In the united states, it is the most common chronic liver disease with an estimated 8 million to 1 million people affected. Non-alcoholic fatty liver disease is a general term for a range of liver diseases affecting those who have little or no alcohol. As the name suggests, the main feature of non-alcoholic fatty liver disease is that there is too much fat stored in hepatocytes. It is normal for the liver to contain some fat. However, if more than 5% -10% of the liver weight is fat, the condition is called fatty liver (steatosis). NAFLD is closely associated with the characteristics of metabolic syndrome, including obesity, insulin resistance, type 2 diabetes, and dyslipidemia; it is considered to be the hepatic manifestation of this syndrome.

NAFLD in children is currently the predominant form of liver disease in children. Studies have shown that abdominal obesity and insulin resistance are considered key factors in the development of NAFLD. As obesity is becoming an increasingly common problem worldwide, the prevalence of NAFLD is increasing. The only treatment that appears to be truly effective in pediatric NAFLD is weight loss.

The more severe form of NAFLD is known as nonalcoholic steatohepatitis (NASH). NASH causes the liver to swell and become damaged. NASH tends to occur in people who are overweight or obese or who have diabetes, high cholesterol or high triglycerides or inflammatory conditions. NASH is a potentially serious form of disease characterized by ballooning of hepatocytes and inflammation of the liver, which may lead to scarring and irreversible injury. This damage is similar to that caused by heavy alcohol use. Both macroscopically and microscopically, NASH is characterized by lobular and/or portal phlebitis, varying degrees of fibrosis, hepatocyte death, and pathological angiogenesis. In the most severe cases, NASH can progress to cirrhosis, hepatocellular carcinoma, and liver failure. NAFLD and NASH are currently being treated, for example, by diet, insulin resistance therapy or vitamin administration, such as vitamin E or D. Unfortunately, no drug is currently approved for the treatment of NAFLD or NASH.

The NAFLD Activity Score (NAS) can be calculated according to the criteria of Kleiner (Kleiner DE. et al Hepatology, 2005; 41: 1313). NAS scores 0-2 were considered undiagnosed, ambiguous or positive for NASH, NAS scores 3-4 were considered undiagnosed, ambiguous or positive for NASH, while NAS scores 5-8 were considered essentially undiagnosed. The therapeutic effect of NASH includes regression, stabilization or reduction of disease progression. Sequential liver biopsies from patients who are likely to have NASH can be used to assess changes in NAS scores and as indicators of changes in disease status. An increasing score indicates progression, a constant score indicates stabilization, and a decreasing score indicates regression of NASH. In a control clinical trial, differences in NAS scores between placebo and test sample treatment groups, typically assessed over a period of 6 months to two years, can indicate treatment efficacy, even if both groups have progressed. Regulatory agencies often require a clear point difference to prove meaningful changes in NASH.

Fibrinogen (factor I) is a mammalian glycoprotein that plays a role in the formation of blood clots. Fibrinogen is converted to fibrin by thrombin during clot formation. Fibrinogen is synthesized in liver hepatocytes. A variety of diseases are associated with elevated fibrinogen levels and include, but are not limited to, NASH, microvascular disease, peripheral vascular disease, peripheral arterial disease, critical limb ischemia in peripheral arterial occlusive disease, new onset coronary atherosclerosis, decreased survival in patients with cancer such as breast cancer, renal cell carcinoma, prostate cancer. Increased fibrinogen levels are also associated with negative sepsis consequences, diabetes, metabolic syndrome and subacute thyroiditis, plasma triglycerides, obesity, ultrasound intraabdominal fat, diastolic blood pressure, insulin resistance, LDL-cholesterol and smoking. In otherwise healthy patients, the severity of obstructive sleep apnea is also associated with elevated plasma fibrinogen.

Fibrinogen may therefore be a prognostic indicator or blood marker for many diseases and may also be used to effect the onset and progression of a disease state. There is a medical need to reduce fibrinogen in subjects with high levels.

Since treatment options for patients with type IIb hyperlipidemia are limited and because current treatments may increase the risk of serious side effects or may not be well tolerated, additional treatments are needed to safely and effectively treat patients with type IIb hyperlipidemia. In addition, current treatments for NAFLD and NASH are limited, and therefore more treatment options are needed that are safe and effective for treating patients with NAFLD and NASH.

SUMMARY

The present invention meets these needs. We have demonstrated that the combined treatment of patients with type IIb hyperlipidemia with certain doses of gemcabene and low or medium doses of statins, demonstrated a reduction in LDL-C and an unexpected supra-additive reduction in Triglycerides (TG) compared to the effect of each agent alone (unexpected more than additive reduction). Gemcabene does not significantly affect the activity or expression of the major cytochrome P450 enzymes involved in metabolism of the agent. Thus, gemcabene can reduce the need to use high doses of statins, thereby reducing the risk of side effects.

A first aspect of the invention provides a method for treating a subject suffering from type IIb hyperlipidemia comprising administering to the subject gemcabene in combination with a low or medium dose of a statin. In some embodiments of the first aspect of the invention, the gemcabene and statin are administered as a fixed dose combination.

A second aspect of the invention provides a method for reducing liver fat accumulation in a subject comprising administering gemcabene, alone or in combination with a statin. In some embodiments of the second aspect of the invention, the method is a method of treating or preventing nonalcoholic fatty liver disease (NAFLD). Another embodiment of the second aspect is a method of treating or preventing steatosis. In some embodiments, the method is a method of treating or preventing a liver disease, wherein the liver disease is non-alcoholic steatohepatitis (NASH).

A third aspect of the invention provides specific fixed dose combinations of gemcabene and a statin.

A fourth aspect provides a kit for treating a subject having type IIb hyperlipidemia and/or NASH comprising gemcabene, a statin, and instructions for use.

A fifth aspect of the invention provides a method for reducing fibrosis in a patient comprising administering gemcabene, alone or in combination with a statin.

A sixth aspect of the invention provides a method for reducing fibrinogen levels in plasma levels in a subject with elevated plasma fibrinogen levels comprising administering gemcabene, alone or in combination with a statin.

One embodiment of the first aspect is a method for treating a subject with type IIb hyperlipidemia comprising administering to the subject gemcabene in combination with a low or medium dose of a statin.

Another embodiment of the first aspect is a method of treating a subject having type IIb hyperlipidemia, comprising administering to the subject a daily dose of gemcabene of about 50mg to about 750mg and a daily dose of statin of about 1mg to about 60 mg.

One embodiment of the second aspect is a method of reducing liver fat accumulation in a subject comprising administering to a subject in need thereof a daily dose of about 50mg to about 750mg gemcabene, and a daily dose of about 1mg to about 80mg statin.

An embodiment of the fourth aspect of the present invention provides a kit comprising: a fixed dose combination comprising from about 1mg to about 60mg of a statin and from about 300mg to about 600mg of gemcabene; and instructions for use thereof. In some embodiments, the statin is selected from the group consisting of atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin and pitavastatin; or any pharmaceutically acceptable salt thereof. In some embodiments, the statin is atorvastatin.

One embodiment of the fifth aspect is a method for reducing fibrosis in a subject comprising administering to a subject in need thereof a daily dose of about 50mg to about 750mg gemcabene, and administering a daily dose of about 1mg to about 80mg statin.

An embodiment of the sixth aspect of the invention is for reducing fibrinogen levels above 300

A method of plasma fibrinogen levels in a subject at mg/dL comprising administering gemcabene to the subject alone or in combination with a statin. The subject may have a high LDL-C level. The subject may be at risk of developing coronary heart disease or already has one or more cardiac events.

Brief Description of Drawings

The following drawings are provided by way of example and are not intended to limit the scope of the present invention.

Figure 1A is a graph showing the effect of gemcabene on atorvastatin concentration in a crossover study in human subjects, where atorvastatin is administered at 80mg alone (filled circles) and in combination with 300mg of gemcabene (open circles) or in combination with 900mg of gemcabene (filled squares).

Figure 1B is a graph showing the effect of gemcabene on atorvastatin lactone concentration in a crossover study in human subjects when atorvastatin is administered at 80mg alone (filled circles) and in combination with 300mg of gemcabene (open circles) or with 900mg of gemcabene (filled squares).

Figure 1C is a graph showing the effect of gemcabene on the concentration of ortho-hydroxy atorvastatin when atorvastatin is administered at 80mg alone (filled circles) and in combination with 300mg of gemcabene (open circles) or with 900mg of gemcabene (filled squares) in a crossover study in human subjects.

Figure 1D is a graph showing the effect of gemcabene on the concentration of ortho-hydroxy atorvastatin lactone in a crossover study in human subjects when atorvastatin is administered at 80mg alone (filled circles) and in combination with 300mg gemcabene (open circles) or with 900mg gemcabene (filled squares).

Figure 1E is a graph showing the effect of gemcabene on the concentration of atorvastatin hydroxy when atorvastatin is administered at 80mg alone (filled circles) and in combination with 300mg of gemcabene (open circles) or with 900mg of gemcabene (filled squares) in a crossover study in human subjects.

Figure 1F is a graph showing the effect of gemcabene on the concentration of hydroxy atorvastatin lactone in a crossover study in human subjects when atorvastatin is administered at 80mg alone (filled circles) and in combination with 300mg gemcabene (open circles) or with 900mg gemcabene (filled squares).

Figure 1G is a graph showing the effect of gemcabene on the pharmacokinetics of atorvastatin administered alone at 80mg (filled circles) and in combination with 900mg of gemcabene (open circles) as measured by activity at HMG-Co a reductase inhibitor concentrations in a crossover study in human subjects.

FIG. 1H is a graph showing the effect of gemcabene on the pharmacokinetics of simvastatin administered alone at 80mg (filled circles) and in combination with 900mg gemcabene (open circles) in a crossover study in human subjects, as measured by activity at HMG-Co A reductase inhibitor concentrations.

Figures 2A and 2B are histograms showing that placebo is being administered according to the study described in example 4; 10mg atorvastatin (statin); 300mg, 600mg or 900mg of gemcabene (gem); or median percent (2A) and average percent (2B) changes in LDL, baseline triglyceride, and baseline ApoB levels from baseline in type IIb patients of 10mg atorvastatin and 300mg, 600mg, or 900mg gemcabene.

Figures 2C and 2D are histograms showing that placebo was being administered according to the study described in example 4; 40mg atorvastatin (statin); 300mg, 600mg or 900mg of gemcabene (gem); or median percent (2C) and average percent (2D) changes in LDL, baseline triglyceride, and baseline ApoB levels from baseline in type IIb patients of 40mg of atorvastatin and 300mg, 600mg, or 900mg of gemcabene.

Figures 2E and 2F are histograms showing that placebo was being administered according to the study described in example 4; 80mg atorvastatin (statin); 300mg, 600mg or 900mg of gemcabene (gem); or median percent (2E) and average percent (2F) changes in LDL, baseline triglyceride, and baseline Apo B levels from baseline in type lib patients of 80mg atorvastatin and 300mg, 600mg, or 900mg gemcabene.

FIG. 3 is a bar graph showing the effect of 100mg/kg gemcabene (Gem) and 3mg/kg simvastatin (Simva) on plasma cholesterol, liver cholesterol and liver triglyceride synthesis.

FIG. 4 is a graph showing the mean body weight change of the diabetic mouse NASH model as a function of the days after the start of treatment and the administered dose.

Fig. 5A is a graph showing body weight of the diabetic mouse NASH model on the day of treatment termination.

Fig. 5B is a graph showing liver weight of the diabetic mouse NASH model on the day of treatment termination.

Fig. 5C is a graph showing the liver to body weight ratio of the diabetic mouse NASH model on the day of treatment termination.

Fig. 6A is a graph showing fasting whole blood glucose levels for the NASH model in diabetic mice 3 days prior to termination and after 8 hours of fasting.

Fig. 6B is a graph showing fasting blood glucose levels of the NASH model in diabetic mice 3 days before termination and after 8 hours of fasting.

FIG. 7A is a graph showing the whole blood glucose level of the NASH model in diabetic mice at termination.

FIG. 7B is a graph showing plasma alanine Aminotransferase (ALT) levels at termination in the diabetic mouse NASH model.

FIG. 7C is a graph showing plasma aspartate transaminase (AST) levels at termination in the diabetic mouse NASH model.

Fig. 7D is a graph showing plasma α lipoic Acid (ALP) levels of the NASH model in diabetic mice at termination.

FIG. 7E is a graph showing plasma Gamma Glutamyl Transferase (GGT) levels at termination in the diabetic mouse NASH model.

FIG. 7F is a graph showing Blood Urea Nitrogen (BUN) levels at termination in the NASH model in diabetic mice.

Fig. 7G is a graph showing plasma creatinine levels in the mouse NASH model of diabetes at termination.

Fig. 7H is a graph showing plasma total bilirubin levels at the termination time of the diabetic mouse NASH model.

FIG. 7I is a graph showing plasma ketone body levels at termination in the diabetic mouse NASH model.

Fig. 7J is a graph showing hepatic triglyceride levels at termination in the diabetic mouse NASH model.

Fig. 8 is a graph showing the non-alcoholic fatty liver disease (NAFLD) score of the diabetic mouse NASH model at termination.

Fig. 9A is a graph showing the steatosis score of the diabetic mouse NASH model at termination.

Fig. 9B is a graph showing the lobular inflammation score of the diabetic mouse NASH model at termination.

Fig. 9C is a graph showing the balloonlike degeneration score of the diabetic mouse NASH model at termination.

Fig. 10 is a graph showing the area of fibrosis (% sirius red positive area) of the diabetic mouse NASH model at termination.

FIG. 11A is a bar graph showing hepatic triglyceride levels in male Sprague-Dawley rats after treatment with gemfibrozil or gemcabene.

FIG. 11B is a bar graph showing liver unesterified cholesterol levels in male Sprague-Dawley rats after treatment with gemfibrozil or gemcabene.

FIG. 12 is a bar graph showing plasma fibrinogen levels in male Sprague-Dawley rats after treatment with gemfibrozil or gemcabene.

Detailed description of the invention

Definition of

"API" is an abbreviation for active pharmaceutical ingredient.

Statins are a class of drugs that inhibit HMG-CoA reductase and are generally known to lower LDL cholesterol in patients. Examples of statins include atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin and pitavastatin.

As used herein, a "type IIb hyperlipidemia" or "type IIb" patient population refers to a patient population having a fasting LDL cholesterol plasma level of greater than or equal to 130mg/dL and a fasting triglyceride plasma level of greater than or equal to 150 mg/dL. Unless otherwise specifically indicated, reference to LDL-C, triglyceride, or ApoB levels is a fasting level. Type IIb hyperlipidemia is also known as type IIb hyperlipoproteinemia. In some references, type IIb hyperlipidemia is referred to as mixed dyslipidemia or is described as a subgroup of mixed dyslipidemia.

As used herein, the term "gemcabene" refers to the compound 6,6' -oxybis (2, 2-dimethylhexanoic acid) having the structure

The gemcabene calcium is monocalcium salt of gemcabene. Calcium gemcabene may be used interchangeably with gemcabene.

As used herein: "steatosis" is interchangeable with "fatty liver," which is the accumulation of fat in the liver.

When referring to the dosage amount and dose, the dosage amount or dose is calculated from the weight of the API. In some embodiments, the API may be administered as a pharmaceutically acceptable salt. When the API is administered in salt form, the dosage is still calculated on the basis of the API. For example, reference to a dose of 80mg atorvastatin calcium means a dose of atorvastatin calcium equivalent to 80mg atorvastatin, and reference to a dose of 300mg gemcabene calcium means a dose of gemcabene calcium equivalent to 300mg gemcabene.

As used herein, the term "single dose formulation" or "fixed dose combination" refers to a pharmaceutical composition in its commercially available form that is formulated using a mixture of two or more APIs and one or more excipients, as well as other optionally non-reusable materials that may not be considered ingredients or packaging (e.g., capsule shells). As used herein, the terms "single dose formulation" and "fixed dose combination" are used interchangeably. Common single dose formulations include pills, tablets or capsules.

As used herein, "subject" and "patient" are used interchangeably. The subject may be a mammal, which may be, for example, a human, including adult, juvenile, and pediatric subjects.

"steatosis" and "hepatic steatosis" are used interchangeably herein.

"Blood plasma" and "plasma" are used interchangeably herein.

The following table provides the dosage categories for a number of statins used herein.

TABLE 1

The present invention provides methods for treating a subject having type IIb hyperlipidemia. As described in further detail in example 3, a double-blind randomized placebo-controlled dose range study over an 8-week period was conducted to assess the efficacy and safety of gemcabene treatment of hypercholesterolemic patients administered as monotherapy or in combination with atorvastatin. The primary objective was to evaluate the low density lipoprotein cholesterol (LDL-C) lowering efficacy and dose response of gemcabene 300, 600 and 900 mg/day administered to hypercholesterolemic patients (friedrickson types IIa and IIb) as monotherapy or in combination with atorvastatin 10, 40 and 80 mg/day. A secondary objective was to evaluate the regulation of hypersensitive c-reactive protein (hsCRP), high density lipoprotein cholesterol (HDL), and Triglycerides (TG) and apolipoprotein b (apob) by gemcabene.

Subjects were randomly assigned to receive placebo, a medicament as monotherapy or a combination of medicaments at different dose levels for 8 weeks. Before and at the end of the treatment period, safety and lipid variables, including plasma triglyceride, LDL-C and apoB levels, were assessed.

Subgroup analysis of LDL-C and TG in subjects with LDL-C levels ≧ 130mg/dL and triglyceride levels ≧ 150mg/dL (type IIb) showed unexpected reductions in triglycerides in patients administered less than the maximal dose (80mg) of atorvastatin plus gemcabene (300, 600, or 900 mg). The reduction in triglycerides with the combination therapy was much greater than the reduction with atorvastatin or gemcabene monotherapy in the other doses tested. Furthermore, these combinations further resulted in a reduction in LDL-C and apo B compared to atorvastatin or gemcabene administered alone.

The discovery that the use of low to moderate doses of statins in combination with gemcabene resulted in a surprising reduction in TG in this group of subjects potentially provided a safety advantage.

Cytochrome P450 enzymes mediate drug metabolism in humans. For example, some statins and fibrates use or activate the cytochrome P4503a4 isoform as part of their catabolic processes. When administered together, some statins and fibrates compete for the cytochrome P4503a4 isoform, resulting in drug-drug interactions that affect the level of each agent in the plasma.

As an example, statins sold on the market as Baycol are removed from the market after severe drug-drug interaction (DDI) with gemfibrozil (a fibrate) that leads to rhabdomyolysis and patient death.

As further described in example 1 below, no evidence of significant inhibition was observed for any cytochrome P450 isoforms tested at concentrations up to 1500 μ M, indicating that metabolic-based clinical interactions between gemcabene and statins are highly unlikely to occur at therapeutic concentrations of gemcabene.

In addition, where gemcabene monotherapy is administered in a single dose of 1500mg and multiple doses of 900mg, the number of musculoskeletal adverse events is similar to or less than placebo, while statin monotherapy shows an increase in musculoskeletal adverse events compared to placebo. As shown in table 2, co-administration of gemcabene and statins did not increase the musculoskeletal adverse effects caused by statin alone.

TABLE 2

"n" is the number of patients in the group.

No ═ the number of musculoskeletal adverse events and% > -the percentage of patients in a particular group who experienced a musculoskeletal adverse event.

Detailed description of the preferred embodiments

A first aspect of the invention provides a method for treating a subject suffering from type IIb hyperlipidemia comprising administering to the subject gemcabene in combination with a low or medium dose of a statin. In some embodiments of the invention, gemcabene and a statin are administered as a fixed dose combination. A third aspect of the invention provides specific fixed dose combinations of gemcabene and a statin. A fourth aspect provides a kit for treating a subject suffering from type IIb hyperlipidemia and/or NASH. A fifth aspect of the invention provides a method for reducing fibrosis in a patient comprising administering gemcabene, alone or in combination with a statin.

Gemcabene is typically administered as the monocalcium salt (calcium gemcabene).

One embodiment of the first aspect is a method for treating a subject with type IIb hyperlipidemia comprising administering to the subject gemcabene in combination with a low or moderate intensity dose of a statin.

Another embodiment is a method of treating a subject having type IIb hyperlipidemia, comprising administering to the subject a daily dose of about 50mg to about 750mg of gemcabene and a daily dose of about 1mg to about 60mg of a statin.

Yet another embodiment is a method of treating a subject having type IIb hyperlipidemia comprising administering to the subject a daily dose of about 50mg to about 900mg of gemcabene and a statin, wherein the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin or pitavastatin. Another embodiment is a method of treating a subject having type IIb hyperlipidemia comprising administering to the subject a daily dose of about 150mg to about 600mg of a gemcabene and a statin, wherein the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin. Another embodiment is a method of treating a subject having type IIb hyperlipidemia comprising administering to the subject a daily dose of about 150mg to about 450mg of gemcabene and a statin, wherein the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin.

Yet another embodiment is a method of treating a subject having type IIb hyperlipidemia, comprising administering to the subject a daily dose of gemcabene.

Yet another embodiment is a method of treating a subject having type IIb hyperlipidemia, comprising administering to the subject a daily dose of about 50mg to about 600mg, or about 150mg to about 450mg, or about 150mg to about 300mg of a daily dose of gemcabene and a statin, wherein the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin or pitavastatin, and:

a. the statin is atorvastatin having a daily dose of about 10mg to about 60mg or about 5mg to about 60 mg;

b. the statin is rosuvastatin in a daily dose of about 5mg to about 20mg or about 2.5mg to about 30 mg;

c. the statin is simvastatin in a daily dose of about 10mg to about 40mg or about 5mg to about 60 mg;

d. the statin is pravastatin and the daily dose of pravastatin is from about 10mg to about 80mg or from about 5mg to about 60 mg;

e. the statin is lovastatin in a daily dose of about 20mg to about 40mg or about 10mg to about 60 mg;

f. the statin is fluvastatin and the daily dose of fluvastatin is from about 20mg to about 80mg or from about 1mg to about 60 mg; or

g. The statin is pitavastatin and the daily dose of pitavastatin is from about 1mg to about 4 mg.

In any of the above embodiments, the daily dose of gemcabene is from about 50mg to about 600 mg. In some of the above embodiments, the daily dose of gemcabene is 50mg, 75mg, 100mg, 150mg, 300mg, 400mg, 450mg, 500mg, or 600 mg.

In some embodiments of the method of treating a subject with type IIb hyperlipidemia, the daily dose of atorvastatin is from about 10mg to about 40 mg.

In some embodiments of the methods of treating a subject with type IIb hyperlipidemia, the daily dose of rosuvastatin is from about 10 to about 20 mg.

In some embodiments of the methods of treating a subject with type IIb hyperlipidemia, the daily dose of simvastatin is from about 10mg to about 20 mg.

In some embodiments of the methods of treating a subject with type IIb hyperlipidemia, the daily dose of pravastatin is from about 10mg to about 40 mg.

In some embodiments of the method of treating a subject with type IIb hyperlipidemia, the daily dose of lovastatin is about 40 mg.

In some embodiments of the method of treating a subject with type IIb hyperlipidemia, the daily dose of fluvastatin is from about 20mg to about 40 mg.

In some embodiments of the method of treating a subject with type IIb hyperlipidemia, the daily dose of pitavastatin is from about 1mg to about 3 mg.

In any embodiment of the method of treating a subject having type IIb hyperlipidemia, the statin and gemcabene can be administered as a fixed dose combination.

Some embodiments of the invention patients have Familial Combined Hyperlipidemia (FCHL).

In some embodiments of the methods of treating a subject with type IIb hyperlipidemia, the subject's plasma triglyceride levels are reduced to less than 150mg/dl within 8 weeks of administration of gemcabene and a statin.

In other embodiments of the method of treating a subject with type IIb hyperlipidemia, the subject's plasma LDL cholesterol level is reduced to less than 130mg/dl within 8 weeks of administration of gemcabene and a statin.

In other embodiments of the method of treating a subject with type IIb hyperlipidemia, the subject's plasma triglyceride level is reduced to less than 150mg/dl and the subject's LDL cholesterol level is reduced to less than 130mg/dl within 8 weeks of administration of gemcabene and a statin.

In some embodiments of the methods of treating a subject with type IIb hyperlipidemia, the subject's HDL cholesterol level is increased following treatment.

In some embodiments of the methods of treating a subject with type IIb hyperlipidemia, the subject is at a reduced risk of myopathy as compared to the risk of administering a high dose statin alone.

In some embodiments of the methods of treating a subject with type IIb hyperlipidemia, the subject is at a reduced risk of myositis as compared to the risk of administering a high dose statin alone.

In some embodiments of the methods of treating a subject with type IIb hyperlipidemia, the subject is at a reduced risk of rhabdomyolysis compared to the risk of administering a high dose statin alone.

In any embodiment of the methods of the invention, the subject may be administered an additional cholesterol lowering agent. In some embodiments, the additional cholesterol lowering agent is a cholesterol absorption inhibitor. In some embodiments, the cholesterol absorption inhibitor is ezetimibe. In some embodiments, the cholesterol-lowering agent is a PCKS9 inhibitor.

In some embodiments of the methods of treating a subject with type IIb hyperlipidemia, the subject is at reduced risk of having a primary cardiovascular event.

In some embodiments of the methods of treating a subject with type IIb hyperlipidemia, the subject is at reduced risk of having a secondary cardiovascular event. Another embodiment is a method for treating a patient having type IIb hyperlipidemia comprising administering to the subject a daily dose of gemcabene of about 150mg and a daily dose of atorvastatin selected from 10mg, 20mg, 30mg, 40mg, 50mg or 60 mg.

Yet another embodiment is a method for treating a patient with type IIb hyperlipidemia comprising administering to the subject a daily dose of about 300mg of gemcabene and a daily dose of atorvastatin selected from 10mg, 20mg, 30mg, 40mg, 50mg or 60 mg.

Yet another embodiment is a method for treating a patient with type IIb hyperlipidemia comprising administering to the subject a daily dose of gemcabene of about 450mg and a daily dose of atorvastatin selected from 10mg, 20mg, 30mg, 40mg, 50mg or 60 mg.

Another embodiment is a method for treating a patient having type IIb hyperlipidemia comprising administering to the subject a daily dose of about 600mg gemcabene and a daily dose selected from 10mg, 20mg, 30mg, 40mg, 50mg or 60mg of atorvastatin.

A second aspect of the invention provides a method of reducing liver fat mass or liver fat accumulation in a subject comprising administering gemcabene, alone or in combination with a statin. In some embodiments of the second aspect, the method is a method of treating a subject to reduce or prevent steatosis, the method comprising administering gemcabene, alone or in combination with a statin. One embodiment of the second aspect is a method of treating a fatty liver disease in a subject comprising administering gemcabene, alone or in combination with a statin. Another embodiment is a method of treating liver disease, wherein the liver disease is non-alcoholic fatty liver disease (NAFLD). In some embodiments of the second aspect, the method is a method of treating a subject to prevent or reduce the rate of progression of a liver disease. In some embodiments, the liver disease is non-alcoholic liver steatosis (NASH). In some embodiments, the liver disease is alcoholic liver steatosis. In some embodiments of the second aspect of the invention, the subject has type IIb hyperlipidemia. In some embodiments, the patient has FCHL. In any embodiment of the second aspect, the subject may have a risk factor for developing fatty liver (steatosis), wherein the risk factor is that the subject has metabolic syndrome, type 2 diabetes, impaired glucose tolerance, obesity, dyslipidemia, hepatitis b, hepatitis c, HIV infection or metabolic disorders such as wilson's disease, glycogen storage disease or galactosemia. In some embodiments, the patient has diabetes. In some embodiments, the patient has an inflammatory condition. In some embodiments, the patient's body mass index is elevated above that which is normal for gender, age, and height.

In some embodiments of the second aspect, the method is a method of reducing liver fat accumulation in a patient by administering gemcabene to the patient as monotherapy. In some embodiments, gemcabene is administered with one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents is a statin. In some embodiments, the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin or pitavastatin.

In any embodiment of the second aspect, the method of preventing or reducing liver fat accumulation in a subject comprises administering to a subject in need thereof a daily dose of about 50mg to about 900mg gemcabene.

Another embodiment of the second aspect is a method of preventing or reducing liver fat accumulation in a subject comprising administering to a subject in need thereof a daily dose of about 50mg to about 900mg of gemcabene, and a daily dose of about 1mg to about 80mg of a statin.

Another embodiment of the second aspect is a method of reducing liver fat accumulation in a subject comprising administering to a subject in need thereof a combination of gemcabene and a statin, wherein the daily dose of gemcabene administered to the subject is from about 50mg to about 900mg per day and the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin or pitavastatin.

In any embodiment of the method of preventing or reducing liver fat accumulation in a subject, the daily dose of gemcabene is from about 50mg to about 900 mg.

In any embodiment of the method of reducing liver fat accumulation in a subject, the daily dose of gemcabene is 150mg, 300mg, 450mg, or 600 mg.

In any embodiment of the method for reducing liver fat accumulation in a subject, the daily dose of atorvastatin is from about 10mg to about 80 mg.

In any embodiment of the method for reducing liver fat accumulation in a subject, the daily dose of rosuvastatin is about 5 to about 40 mg.

In any embodiment of the method for reducing liver fat accumulation, the simvastatin is about 10mg to about 20 mg.

In any embodiment of the method for reducing liver fat accumulation, the daily dose of pravastatin is from about 10mg to about 40 mg.

In any embodiment of the method for reducing liver fat accumulation, the daily dose of lovastatin is about 20 to about 40 mg.

In any embodiment of the method for reducing liver fat accumulation, the daily dose of fluvastatin is from about 20mg to about 40 mg.

In any embodiment of the method for reducing liver fat accumulation, the daily dose of pitavastatin is about 1mg to about 3 mg.

In any embodiment of the method for reducing liver fat accumulation, the statin and gemcabene may be administered as a fixed dose combination.

In some embodiments of the methods for reducing liver fat accumulation, the subject is at reduced risk of developing a liver disease.

In some embodiments of the methods for reducing liver fat accumulation, the subject has liver disease.

In some embodiments of the method for reducing liver fat accumulation, the fat is triglyceride.

In some embodiments, the liver disease is non-alcoholic steatohepatitis (NASH).

In some embodiments, the liver disease is non-alcoholic fatty liver disease (NAFLD).

In some embodiments, the liver disease is liver fibrosis.

In some embodiments, the liver disease is inflammation of the liver.

In some embodiments, the liver disease is cirrhosis.

In one embodiment of the second aspect of the invention, a daily dose of about 75mg of gemcabene and a daily dose of 1 to 80mg of a statin is administered to the subject.

In a specific embodiment of the second aspect, a daily dose of about 150mg of gemcabene and a daily dose selected from 10mg, 20mg, 30mg, 40mg, 50mg, 60mg or 80mg of atorvastatin is administered to the subject.

In another specific embodiment of the second aspect, a daily dose of about 300mg of gemcabene and a daily dose selected from 10mg, 20mg, 30mg, 40mg, 50mg or 60mg of atorvastatin is administered to the subject.

In a specific embodiment of the second aspect, a daily dose of about 450mg of gemcabene and a daily dose selected from 10mg, 20mg, 30mg, 40mg, 50mg or 60mg of atorvastatin is administered to the subject.

In another specific embodiment of the second aspect, a daily dose of about 600mg of gemcabene and a daily dose selected from 10mg, 20mg, 30mg, 40mg, 50mg or 60mg of atorvastatin is administered to the subject.

In another specific embodiment of the second aspect, a daily dose of about 900mg of gemcabene and a daily dose selected from 10mg, 20mg, 30mg, 40mg, 50mg or 60mg of atorvastatin is administered to the subject.

In a preferred embodiment of the second aspect of the invention, a daily dose of about 450mg of gemcabene and a daily dose selected from 10mg of atorvastatin is administered to the subject. In another preferred embodiment, a daily dose of about 450mg of gemcabene and a selected daily dose of 20mg of atorvastatin is administered to the subject. In another preferred embodiment, a daily dose of about 450mg of gemcabene and a daily dose selected from 40mg of atorvastatin is administered to the subject.

In a preferred embodiment of the second aspect of the invention, a daily dose of about 300mg of gemcabene and a daily dose selected from 10mg of atorvastatin is administered to the subject. In another preferred embodiment, a daily dose of about 300mg of gemcabene and a selected daily dose of 20mg of atorvastatin is administered to the subject. In another preferred embodiment, a daily dose of about 300mg of gemcabene and a daily dose selected from 40mg of atorvastatin is administered to the subject.

In a preferred embodiment of the second aspect of the invention, a daily dose of about 150mg of gemcabene and a daily dose selected from 10mg of atorvastatin is administered to the subject. In another preferred embodiment, a daily dose of about 150mg of gemcabene and a selected daily dose of atorvastatin from 20mg is administered to the subject. In another preferred embodiment, a daily dose of about 150mg of gemcabene and a daily dose selected from 40mg of atorvastatin is administered to the subject.

In some embodiments of the second aspect, the method further comprises administering an additional therapeutic agent, and the additional agent is a drug for treating NASH. In some embodiments, the additional agent is cetuximab, GS-4997, GS-974, GS-0976, INT-47, obeticholic acid, or cenicriviroc.

In embodiment a of the second aspect, the invention includes a method for treating or preventing steatosis, NAFLD or NASH comprising administering to a subject in need thereof an effective amount of a compound of formula (I):

or a pharmaceutically acceptable salt or hydrate, wherein:

(a) each occurrence of m is independently an integer ranging from 0 to 5;

(b) each occurrence of n is independently an integer in the range of 3 to 7;

(c) x is- (CH)₂)_z-、–O-、–CH(OH)-、CH(CH₂OH) -, -NH-or-S-, wherein z is an integer from 0 to 4;

(d) each occurrence of R¹And R²Independently is (C)₁-C₆) Alkyl, (C)₂-C₆) Alkenyl, (C)₂-C₆) Alkynyl, phenyl, benzyl, or R¹And R²With them all attachedTogether with the carbon to form (C)₃-C₇) A cycloalkyl group;

(e) each occurrence of R¹¹And R¹²Together with the carbon to which they are both attached to form (C)₃-C₇) A cycloalkyl group;

(f) each occurrence of Y¹And Y²Independently is (C)₁-C₆) Alkyl, OH, COOH, COOR³、SO₃H、

Wherein:

(i)R³is (C)₁-C₆) Alkyl, (C)₂-C₆) Alkenyl, (C)₂-C₆) Alkynyl, phenyl or benzyl and unsubstituted or substituted by one or more halogen radicals, OH, (C)₁-C₆) An alkoxy group or a phenyl group, or a substituted group,

(ii) each occurrence of R⁴Independently H, (C)₁-C₆) Alkyl, (C)₂-C₆) Alkenyl, (C)₂-C₆) Alkynyl and unsubstituted or substituted by one or two halogen radicals, OH, C₁-C₆Alkoxy or phenyl group substitution; and

(iii) each occurrence of R⁵Independently H, (C)₁-C₆) Alkyl, (C)₂-C₆) Alkenyl, (C)₂-C₆) Alkynyl.

In exemplary compounds of formula (I), each occurrence of Y is independently OH, COOR³Or COOH.

Other compounds of formula (I) are those wherein m is 0.

Other compounds of formula (I) are those wherein m is 1.

Other compounds of formula (I) are those wherein n is 4.

Other compounds of formula (I) are those wherein n is 5.

Other compounds of formula (I) are those wherein z is 0.

Other compounds of formula (I) are those wherein z is 1.

Further compounds of the formula (I) are those in which Y¹And Y²Each independently is (C)₁-C₆) Those of alkyl groups.

Further compounds of the formula (I) are those in which Y¹And Y²Those compounds each being methyl.

Other compounds of formula (I) are those in which R is present at each occurrence¹And R²Together with the carbon to which they are both attached to form (C)³-C⁷) Those of cycloalkyl groups.

Embodiment B of the second aspect of the invention is a method of treating or preventing steatosis, NAFLD or NASH comprising administering to a subject in need thereof an effective amount of a compound of formula (II):

or a pharmaceutically acceptable salt or hydrate, wherein:

(a) each occurrence of m is independently an integer ranging from 0 to 5;

(b) each occurrence of n is independently an integer in the range of 3 to 7;

(d) each occurrence of R¹And R²Independently is (C)₁-C₆) Alkyl, (C)₂-C₆) Alkenyl, (C)₂-C₆) Alkynyl, phenyl, benzyl, or R¹And R²Together with the carbon to which they are both attached to form (C)₃-C₇) A cycloalkyl group;

Wherein:

(ii) each occurrence of R⁴Independently H, (C)₁-C₆) Alkyl, (C)₂-C₆) Alkenyl, (C)₂-C₆) Alkynyl and notSubstituted or by one or two halogen radicals, OH, C₁-C₆Alkoxy or phenyl group substitution; and (iii) each occurrence of R⁵Independently H, (C)₁-C₆) Alkyl, (C)₂-C₆) Alkenyl, (C)₂-C₆) Alkynyl.

In exemplary compounds of formula (II), each occurrence of Y is independently OH, COOR³Or COOH.

Other compounds of formula (II) are those wherein n is 4.

Other compounds of formula (II) are those wherein n is 5.

Other compounds of formula (II) are those wherein z is 0.

Other compounds of formula (II) are those wherein z is 1.

Further compounds of the formula (II) are those in which Y¹And Y²Each independently is (C)₁-C₆) Those of alkyl groups.

Further compounds of the formula (II) are those in which Y¹And Y²Those compounds each being methyl.

Other compounds of formula (II) are those in which R is present at each occurrence¹And R²Together with the carbon to which they are both attached to form (C)³-C⁷) Those of cycloalkyl groups.

In one embodiment according to embodiment B, the compound is cyprinic acid.

In one embodiment according to embodiment B, the compound has the structure:

or a pharmaceutically acceptable salt or hydrate thereof.

In another embodiment according to embodiment B, the compound is gemcabene or a pharmaceutically acceptable salt thereof.

In another embodiment according to embodiment B, the compound is the mono-calcium salt of gemcabene.

In yet another embodiment according to embodiment B, the compound is a hydrate of the monocalcium salt of gemcabene.

In another embodiment according to embodiment B, the compound or pharmaceutically acceptable salt thereof is present in a composition further comprising a pharmaceutically acceptable vehicle or carrier.

In another embodiment according to embodiment B, the composition is formulated for oral administration.

In yet another embodiment according to embodiment B, the composition is in the form of a tablet or capsule.

In one embodiment according to embodiment B, the compound or pharmaceutically acceptable salt thereof is present in the composition in an amount from about 50mg to about 900 mg.

In one embodiment according to embodiment B, the compound or pharmaceutically acceptable salt thereof is present in the composition in an amount from about 1mg to about 1,000 mg.

A third aspect of the invention is a fixed dose combination of gemcabene and a statin.

Fixed dose combinations of the statins gagemcabene are useful in the prevention and treatment of subjects suffering from one or more of: type IIb hyperlipidemia, high liver fat, NAFLD or NASH, and complications associated with these conditions and other conditions requiring lowering LDL-C and/or triglyceride levels. By using a fixed dose combination, the release rate of each component of the dose can be controlled. Because statins, particularly high-dose statins, can cause safety-related complications, modulation and slow release of statins is beneficial and can mitigate the skeletal muscle safety risks posed by high-dose statins. Fixed dose combinations may also be convenient to administer, and reduce the risk of incorrect dosing when administering multiple medications, and may increase compliance.

The fixed dose combination of the present disclosure comprises a pharmaceutical composition comprising gemcabene and a statin in specific amounts. The fixed dose combination of the present invention provides the same dose of API as the dose of API administered separately as described herein.

One embodiment of the third aspect of the invention is a fixed dose combination comprising gemcabene in an amount from about 50mg to about 750mg and a statin in an amount from about 1mg to about 60 mg.

In one embodiment of the fixed dose combination, wherein the amount of gemcabene is from about 150mg to about 600mg and the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin or pitavastatin, wherein:

a. the amount of atorvastatin is from about 10mg to about 60 mg;

b. the amount of rosuvastatin is from about 5mg to about 20 mg;

c. simvastatin in an amount from about 10mg to about 40 mg;

d. the amount of pravastatin is from about 10mg to about 80 mg;

e. the amount of lovastatin is about 20mg to about 40 mg;

f. the amount of fluvastatin is from about 20mg to about 80 mg; or

g. The amount of pitavastatin is from about 1mg to about 4 mg.

In some embodiments of the third aspect of the present invention, the amount of gemcabene is from about 300mg to about 600 mg.

In some embodiments, the amount of gemcabene is 300mg or 600 mg.

In some embodiments, the amount of atorvastatin is from about 10mg to about 40 mg.

In other embodiments, the amount of rosuvastatin is from about 10 to about 20 mg.

In other embodiments, the amount of simvastatin is from about 10mg to about 20 mg.

In some embodiments, the amount of pravastatin is from about 10mg to about 20 mg.

In other embodiments, the amount of lovastatin is about 40 mg.

In other embodiments, the amount of fluvastatin is from about 20mg to about 40 mg.

In other embodiments, the amount of pitavastatin is from about 1mg to about 3 mg.

In some embodiments, the fixed dose combination comprises 300mg gemcabene and 10mg atorvastatin. In some embodiments, the fixed dose combination comprises 300mg gemcabene and 20mg atorvastatin. In some embodiments, the fixed dose combination comprises 300mg gemcabene and 40mg atorvastatin.

In some embodiments, the fixed dose combination comprises 600mg gemcabene and 10mg atorvastatin. In some embodiments, the fixed dose combination comprises 600mg gemcabene and 20mg atorvastatin. In some embodiments, the fixed dose combination comprises 600mg gemcabene and 40mg atorvastatin.

In some embodiments, the fixed dose combination comprises 900mg of gemcabene and 10mg of atorvastatin. In some embodiments, the fixed dose combination comprises 900mg gemcabene and 20mg atorvastatin. In some embodiments, the fixed dose combination comprises 900mg gemcabene and 40mg atorvastatin. In some embodiments, the fixed dose combination is in the form of a tablet.

And (4) preparing the preparation.

The compounds useful in the present invention can be formulated into pharmaceutical compositions and administered to a subject, e.g., a human subject, in a variety of forms suitable for the chosen route of administration, i.e., oral, transdermal and parenteral. Such compositions and methods for their preparation are well known and can be found, for example, in Remington's Pharmaceutical Sciences, 19 th edition (Mack Publishing Company, 1995). For example, a typical formulation of gemcabene is described in U.S. patent No. 5,648,387. In one embodiment, gellan is formulated alone or in combination with statins with common excipients and carriers such as starches, binders, diluents, and the like, and molded into tablets, or encapsulated into gelatin capsules for convenient oral administration.

A fourth aspect of the invention provides a kit comprising a fixed dose combination comprising from about 1mg to about 60mg of a statin and from about 150mg to about 900mg of gemcabene; and instructions for use. In some embodiments, the statin is selected from the group consisting of atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin and pitavastatin; or any pharmaceutically acceptable salt thereof. In some embodiments, the statin is atorvastatin.

A fifth aspect of the invention provides a method for reducing fibrosis in a patient comprising administering gemcabene, alone or in combination with a statin. One embodiment of the fifth aspect is a method of reducing liver fibrosis in a subject in need thereof, comprising administering gemcabene to the subject. Another embodiment of the fifth aspect is a method of reducing liver fibrosis in a subject in need thereof, comprising administering to the subject gemcabene wherein the subject has NASH. In some embodiments of the fifth aspect, the daily dose of gemcabene is from about 50mg to about 900 mg. In another embodiment, the daily dose of gemcabene is from about 150mg to about 600 mg. In yet another embodiment, the daily dose of gemcabene is 150mg, 300mg, 450mg, or 600 mg. Another embodiment of the fifth aspect is a method of reducing liver fibrosis in a subject in need thereof comprising administering to the subject gemcabene in combination with a statin. In various embodiments, the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin or pitavastatin.

A sixth aspect of the invention provides a method for reducing plasma fibrinogen levels in a subject comprising administering gemcabene to the subject. One embodiment of the sixth aspect of the present invention is a method of reducing plasma fibrinogen levels in a subject in need thereof, wherein the subject's fibrinogen levels are greater than 300mg/dL, comprising administering gemcabene to the subject. In another embodiment of the sixth aspect of the present invention, there is provided a method for reducing plasma fibrinogen levels in a subject in need thereof, wherein the subject's fibrinogen levels are greater than 400mg/dL, comprising administering gemcabene to the subject. In some embodiments of the sixth aspect, the method comprises administering to the subject a dose of gemcabene of 50mg to 900 mg. In some embodiments, gemcabene is administered at a dose of 150-600 mg. In some embodiments, the dose of gemcabene is 50, 75, 150, 300, 450, 600, or 900 mg. One embodiment of the sixth aspect is a method of reducing plasma fibrinogen levels in a subject in need thereof, comprising administering to the subject gemcabene in combination with a statin. Another embodiment is a method of reducing plasma fibrinogen levels in a subject comprising administering a statin in combination with gemcabene, wherein the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin or pitavastatin. Another embodiment of the sixth aspect is a method of reducing plasma fibrinogen levels in a subject in need thereof, the method comprising administering to the patient a daily dose of 50mg to 900mg of gemcabene and a daily dose of 1mg to 80mg of a statin. Yet another embodiment is a method of reducing plasma fibrinogen levels in a subject in need thereof comprising administering to the subject a daily dose of 300mg, 600mg, or 900mg of gemcabene and a daily dose of 10mg, 40mg, or 80mg of atorvastatin. In yet another embodiment is a method of reducing plasma fibrinogen levels in a subject in need thereof, the method comprising administering to the subject a daily dose of 600mg of gemcabene and a daily dose of 10mg of atorvastatin.

In some embodiments of the sixth aspect, the subject has a decreased risk of a primary cardiovascular event. In other embodiments of the sixth aspect, the subject has a reduced risk of having a secondary cardiovascular event. In any embodiment of the sixth aspect, the subject may be administered an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an anticoagulant or a lipid modulating agent. In some embodiments, the anticoagulant is aspirin, dabigatran, rivaroxaban, clopidogrel apixaban, thienopyridine, warfarin (coumarin) acemetacin, hydrocinnamatoxin, atromamycin, phenindione, ixabeabaxaban, letabaxaban hirudin, lepirudin, bivalirudin, argatroban, dabigatran, ximegergran, batroxobin, fibrinase, heparin, and vitamin E. In another embodiment of the sixth aspect is a method of reducing plasma fibrinogen levels in a subject in need thereof, the method comprising administering to the subject a daily dose of 50mg to 900mg of gemcabene and a daily dose of 1mg to 80mg of a statin to prophylactically reduce or treat a coagulation complication in a subject having: diabetes, impaired glucose tolerance, metabolic syndrome, obesity, cancer, sepsis, sleep apnea, atrial fibrillation, deep vein thrombosis, stroke, hypercoagulable state, myocardial infarction, pulmonary embolism, restenosis, hypertriglyceridemia, hypertension, NAFLD, NASH, or cardiovascular disease.

Another embodiment of the sixth aspect is a method of reducing plasma fibrinogen in a subject in need thereof to reduce blood coagulation, comprising administering gemcabene to the subject at a daily dose of 50mg to 900 mg. Another embodiment of the sixth aspect is a method of reducing plasma fibrinogen in a subject in need thereof to prophylactically reduce blood coagulation, the method comprising administering gemcabene to the subject at a daily dose of 50mg to 900 mg. In some embodiments, gemcabene is administered in combination with a statin. In some embodiments, the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin or pitavastatin, and the daily dose of the statin is 1mg to 80 mg. One embodiment of the sixth aspect is a method of reducing plasma fibrinogen in a subject in need thereof, comprising administering gemcabene to the subject at a daily dose of 50mg to 900mg to treat peripheral vascular disease, peripheral arterial disease, microvascular disease, peripheral arterial occlusive disease or critical limb ischemia.

Examples

Example 1

Cytochrome P450 enzymes mediate drug and foreign biomass metabolism in the human body.

For example, some statins and fibrates use or activate the cytochrome P4503a4 isoform as part of their catabolic processes. When administered together, statins and fibrates compete for the cytochrome P4503a4 isoform, resulting in drug-drug interactions that affect the level of each agent in the plasma.

To investigate whether gemcabene might have similar drug-drug interactions, 100, 300 and 1500mM gemcabene were investigated for their ability to inhibit the seven major cytochrome P450 enzymes CYP1a2, CYP2a6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3a4, using an isoform-selective marker substrate and a human liver microsome formulation, which mediate drug and xenobiotic metabolism in humans.

A series of probe substrates were used to assess the inhibitory activity of gemcabene on the cytochrome P450-dependent isoform-selective metabolic pathway of the probe substrate. Human livers HLM141, HLM143 and HLM144 were selected from the washington university human liver pool for this study. Microsomes were prepared and assayed for the ability of gemcabene (100, 300 and 1500mM or 30.2, 90.7 and 453.6mg/mL) to inhibit a defined metabolic pathway at its Km for each probe substrate. The only exception is CYP1A2, where a concentration of 0.5mM is used, which is below about 1.5mM Km. The actual concentration of each of the other probes is shown below. Experimental controls consisted of complete microsomal incubation with probe substrate in the absence of gemcabene. Assays were performed in triplicate for control and each different gemcabene concentration. The concentration of gemcabene chosen for the preliminary study was designed to ensure that the microsomal concentration would encompass a range that would equal and significantly exceed the concentration of gemcabene expected to be encountered in vivo. The seven isoforms and metabolic pathways of each probe used to monitor the activity of a particular isoform are listed below.

CYP1a 2: 6-hydroxylation of (R) -warfarin at high concentration (0.5 mM). The reaction was monitored by quantitative gas chromatography/mass spectrometry (GC/MS) using 6-hydroxywarfarin- (d 5-phenyl) as an internal standard.

CYP2a 6: 7-hydroxylation of coumarin (4. mu.M). The reaction was monitored by HPLC using fluorescence detection.

CYP2C 9: 7-hydroxylation of (S) -warfarin (4. mu.M). The reaction was monitored by quantitative GC/MS using 7-hydroxywarfarin- (d 5-phenyl) as internal standard.

CYP2C 19: 4' -hydroxylation of (S) -mefentin (50. mu.M). The reaction was monitored by quantitative GC/MS using mefenton- (d 3-methyl) as internal standard.

CYP2D 6: o-demethylation of dextromethorphan (5. mu.M). The reaction was monitored by High Performance Liquid Chromatography (HPLC) using fluorescence detection.

CYP2E 1: p-nitrocatechol was formed from p-nitrophenol (40. mu.M). The amount of product formed was monitored by HPLC.

CYP3a 4: 10-hydroxylation of (R) -warfarin (0.5 mM). The reaction was monitored by quantitative GC/MS using 10-hydroxywarfarin- (d 5-phenyl) as internal standard.

Gemcabene appears to not significantly inhibit any of the seven human cytochrome P450 isoforms examined at three concentrations (100, 300 and 1500 μ M or 30.2, 90.7 and 453.6 μ g/mL, respectively). Although plasma protein binding is non-linear in humans, the gemcabene concentrations used in this study exceed those used in the binding study by a factor of 4. At the highest concentration used (1500 μ M), there appears to be some marginal (10% -20%) inhibitory activity against CYP2a6, CYP2D6 and CYP2C 9. In contrast, CYP2E1 appeared to be slightly (about 20%) enhanced. These results indicate that a metabolism-based clinical interaction between gemcabene and other drugs whose clearance depends on one of the cytochrome P450 isoforms tested is highly unlikely at therapeutic concentrations of gemcabene.

As shown in Table 3, the IC50 value for the major human hepatocytochrome P450 isoform was greater than 1500. mu.M (453.6. mu.g/ml).

TABLE 3

Example 2

Distribution of radiolabeled gemcabene in male rat tissues.

Absorption, distribution, metabolism and elimination studies in rats administered radiolabeled gemcabene showed that almost all of the drug was distributed in the liver and kidneys, i.e. the organs involved in the metabolism and elimination of gemcabene, with little or no distribution to muscles.

The study used a DNA having a specific activity of 47.3. mu. Ci/mg¹⁴C]Gemcabene. Display of position of label¹⁴C]The structure of gemcabene is shown below.

The dosage solution was prepared by dissolving the labeled drug in 0.45mL ethanol and 4.05mL 0.5% methylcellulose in water. Unlabeled gemcabene was added to produce a 10mg/kg solution. The final specific activity of the dose solution was 10.37. mu. Ci/mg.

Male Wistar rats with an average body weight of 202g were fasted overnight before administration. Administering a single mouth to an animalThe dose of oral (PO) is 10.0mg/kg [ alpha ], [ beta ]¹⁴C]Gemcabene (approximately 21 μ Ci/rat) and two animals were sacrificed by overwhelming halothane anesthesia at each time point 1, 4, 8, 24, 48, 96 or 192 hours post-dose. Cadavers were flash frozen in dry ice/hexane mixtures and prepared for whole-body sectioning and autoradiography. Sections 50 μm thick were air dried at-20 ℃ and applied to either a BioMax scientific imaging film (Eastman Kodak, Rochester, New York) or a phosphorus imaging plate for autoradiographic exposure (Molecular Dynamics, Sunnyvale, California). Carbon-14 standards (American radio label chemicals, st. louis) were included on representative membranes and plates used to calibrate the analyzer. The treated membrane was digitized using an analyzer scanner (Loats INQUIRY image analysis system, Loats Associates, Westminster, Maryland) and the remaining radioactivity in the tissue was determined by quantitative video densitometric analysis of the digital images. The imaging plate was scanned after 1 week of exposure. The resulting electronic image is used for preliminary evaluation of the distribution.

Table 4 shows the residual [14C ] gemcabene radioactivity equivalent in the tissue after a 10mg/kg PO dose.

TABLE 4

The lower limit of the amount was 0.10. mu.g/g.

BLQ-less than quantitative level

Example 3

Potential interaction of statins with gemcabene for atorvastatin pharmacokinetics.

To further investigate the potential interactions between statins and gemcabene, the effect of multiple doses of gemcabene (300 and 900mg once daily [ QD ]) on the steady state pharmacokinetics of atorvastatin (80mg QD) was investigated.

The trial was an open label, multiple dose, 1-sequence, 3-treatment, crossover study of healthy subjects.

20 subjects received the following treatment (treatment 1)80mg atorvastatin QD for 5 days; (treatment 2)80mg atorvastatin QD with 300mg gemcabene QD for 11 days (days 6-16); and (treatment 3)80mg atorvastatin QD with 900mg gemcabene QD for 11 days (days 17-27). The medication is administered orally at approximately the same time for each treatment. Each dose was administered with 8 ounces of water.

Serial blood samples for atorvastatin and atorvastatin metabolite assays were collected for 24 hours after one or more of the doses on days 5, 16 and 27. 3 ml of venous blood was drawn into a blood collection tube containing heparin sodium. Blood samples were drawn before dosing and at 0.33, 0.66, 1, 2, 3, 4, 6, 8, 12 and 24 hours after the dose on day 5, day 16 and day 27. After each collection, the blood samples were centrifuged as soon as possible, and the plasma was separated and stored frozen at 70 ℃ until the atorvastatin concentration was determined.

Administration of 80mg atorvastatin alone or in combination with 300 and 900mg gemcabene was well tolerated by healthy volunteers. Based on a comparison of the Cmax, tmax, AUC (0-24) and t1/2 values for atorvastatin and atorvastatin metabolites, gemcabene had no clinically significant effect on the pharmacokinetics of atorvastatin. The pharmacokinetic data for atorvastatin is shown in table 5 and the pharmacokinetic data for atorvastatin total analyte (atorvastatin plus metabolite) is shown in table 6.

TABLE 5

In addition to determining the effect of gemcabene on atorvastatin pharmacokinetics, the effect of gemcabene on metabolite pharmacokinetics was also determined. Based on AUC (0-24) values, the exposure to atorvastatin total analyte after administration of 80mg atorvastatin with 300 or 900mg gemcabene was similar to the exposure to 80mg atorvastatin alone. The difference in mean AUC (0-24) values was less than 7%. The 90% confidence interval for the ratio of log-transformed test/reference treatment AUC (0-24) values ranged from 80% to 125%, indicating the absence of interaction of gemcabene for overall analyte pharmacokinetics. Similar results were observed when the total analyte concentration was expressed as M, or when only the active analytes atorvastatin, o-hydroxy atorvastatin and p-hydroxy atorvastatin were combined prior to analysis.

TABLE 6

Average steady-state atorvastatin plasma concentration-time curves for atorvastatin plasma concentration, atorvastatin lactone concentration, o-hydroxy atorvastatin lactone, p-hydroxy atorvastatin, and p-hydroxy atorvastatin lactone are graphically shown in figures 1A-1E. The mean steady state atorvastatin plus metabolite plasma concentration-time curve is graphically shown in figure 1G. (80mg atorvastatin alone (filled circles), 80mg atorvastatin with 300mg gemcabene (open circles) and 80mg atorvastatin with 900mg gemcabene (filled squares)).

Effect of gemcabene on simvastatin pharmacokinetics.

Healthy adult male and female subjects were treated with oral simvastatin doses once daily for 15 days and oral simvastatin and gemcabene doses once daily for 15 days, spaced apart by a 4-week washout period.

Blood collection (simvastatin) -10 ml venous blood was collected in EDTA-containing evacuated blood collection tubes 0.5, 1, 2, 3, 4, 6, 8, 12 and 24 hours before and after the simvastatin dose on days 15 and 57. At each time point, plasma was collected and divided into aliquots, one for LC/MS assay and the other for EIA assay. After blood collection, the samples were centrifuged and plasma was separated and stored frozen at-80 ℃ until drug concentration was analyzed.

Blood collection (gemcabene) -5 ml of venous blood were drawn into glass evacuated blood collection tubes containing 72USP units of sodium heparin at 0.5, 1, 2, 3, 4, 8, 12 and 24 hours before and after the gemcabene dose on day 15 or 57. After blood collection, the samples were centrifuged and plasma was separated and stored frozen at-20 ℃ until analysis.

Table 7 provides a summary of the values of the pharmacokinetic parameters for HMG-Co-A reductase inhibitors after administration of 80mg simvastatin (reference) and 900mg gemcabene (CI-1027) alone (tested).

TABLE 7

FIG. 1H shows the average active HMG-Co-A reductase inhibitor concentration after administration of simvastatin (80mg), either alone (filled symbols) or in combination with gemcabene (600mg) (open symbols). The results show that gemcabene reduces Cmax but has no effect on AUC. When simvastatin was dosed at high intensity, little effect on the catabolism of simvastatin or simvastatin metabolites was observed (the concentration and parameter values of the active HMG-Co-A reductase inhibitor are reported in ng-equivalents/mL relative to the standard simvastatin acid used in the assay).

Example 4

The effect of a combination of gemcabene and statins in lowering plasma LDL-C, TG and ApoB levels.

A double-blind randomized placebo-controlled dose range study over a period of 8 weeks was conducted to evaluate the efficacy and safety of gemcabene treatment of hypercholesterolemic patients administered as monotherapy or in combination with atorvastatin. The primary objective was to evaluate the low density lipoprotein cholesterol (LDL-C) lowering efficacy and dose response of gemcabene 300, 600 and 900 mg/day administered to hypercholesterolemic patients (friedrickson types IIa and IIb) as monotherapy or in combination with atorvastatin 10, 40 and 80 mg/day. A secondary objective was to assess the modulatory effects of gemcabene on hypersensitive c-reactive protein (hsCRP), high density lipoprotein cholesterol (HDL), Triglycerides (TG) and apolipoprotein B (apo B).

Subgroup analysis of LDL-C and TG in subjects with LDL-C levels ≧ 130mg/dL and triglyceride levels ≧ 150mg/dL (type IIb) showed supra-additive reductions in triglycerides in patients administered less than 80mg atorvastatin plus gemcabene (300, 600, or 900 mg). The reduction in triglycerides using the combination therapy was much greater than the reduction using atorvastatin or gemcabene monotherapy. In addition, these combinations also resulted in further reductions in LDL-C and apo B.

The study was a parallel group, 4 x 4 factor designed, randomized, double-blind, placebo-controlled, multicenter study in hypercholesterolemic patients. The following table reflects a subset of subjects classified as having type IIb hypercholesterolemia.

Table 8 shows the 4 x 4 factor design used during 8-week double-blind treatment.

TABLE 8

There were 3 phases in the study: (1) carrying out blood fat medical clearance follow-up visit when needed; (2) a trial period; and (3) an 8-week double-blind treatment period. As detailed above (table 6), patients were randomized with the same probability to receive 1 of 16 drug treatments containing different doses of gemcabene and/or atorvastatin and/or placebo. Study medicine was administered orally once a day in the morning (QD). During the 8 week treatment period, patients, field personnel and sponsors were blinded to treatment and plasma lipid levels. "n" in the above table indicates the number of patients in the study with LDL-C ≧ 130mg/dL and TG >150mg/dL at baseline (type IIb patients).

Study medications were dispensed at 4 week intervals in 7 day trays with independent daily dispenses. The patient was instructed to take all tablets in the morning corresponding to the appropriate daily dispense. Gemcabene was provided as 300mg tablets with a matching placebo. Atorvastatin was provided as 10 or 40mg tablets with matched placebo. Study medications were packaged in 7-day blister packs with individual daily dispensers containing 6 tablets per day.

Basal lipid assessments were performed on blood samples collected at each clinic visit. For total cholesterol, LDL-C, HDL-C, and triglycerides, the baseline and percent change from baseline were analyzed using a cross-ANOVA model. The data for the first and second phases were also analyzed using ANOVA models consistent only with treatment effects, respectively, due to potential cycling effects.

The results are shown in fig. 2A to 2F.

The combination of 10mg atorvastatin and 300mg and 600mg gemcabene reduced TG levels by an additional 10.5% and 17.3%, respectively, compared to atorvastatin alone. (median change%) gemcabene at 300, 600 and 900 doses further reduced LDL-C and ApoB compared to 10mg atorvastatin alone.

The combination of 40mg atorvastatin with 300, 600 and 900mg gemcabene further reduced TG levels by 42.6%, 31.7% and 21.8%, respectively, compared to atorvastatin alone. (median Change%)

Example 5

Effect of gemcabene, atorvastatin and acetyl-CoA carboxylase (ACC) inhibitors on cholesterol and triglyceride synthesis in primary rat hepatocyte cultures

All experiments were performed using a method according to Ramharack R. et al J Lipid Res 1995; 36:1294-304 cultures of isolated and cultured primary rat hepatocytes modified (Sprague-Dawley). One day after plating, cells were incubated at the indicated concentrations in triplicate with compounds in DMSO (final concentration of 1%) at 1.0mL per well in 6-well plates of fetal bovine serum removed parenchymal cell culture medium. Cells were incubated at 37 ℃ in 95% O₂/5％CO₂Tissue culture incubators were incubated for 2 hours. At the end of the culture period, the medium was replaced with 1.0mL per well of a labeled medium consisting of a compound at a specified concentration in a parenchymal cell culture medium from which fetal bovine serum was removed and 1-, [ 30 μ Ci ], [1 ], [ 2 ], [ C ] of 30 μ Ci¹⁴C]Acetate (Amersham), and incubated at 37 ℃ for 4 hours in a tissue incubator. At the end of the labeling period, cells were washed with 2.0mL of room temperature D-PBS per well and quenched with 1.0mL of 0.75N HCl. The cells were scraped and transferred to 15X 45mm glass vials (1 dram size). The wells were washed with 1.0mL of methanol and scraped cells were added followed by 2.0mL of chloroform. The vial was capped and vortexed for 5 seconds, and centrifuged at 3,600rpm for 15 minutes in a Beckman GS 6KR centrifuge at room temperature to separate the phases. The bottom chloroform phase was transferred to a new 15x 45mm glass vial and dried under nitrogen in a ReactiVap evaporator at 37 ℃. The vial was cooled to room temperature and the sample was resuspended in 130 μ L of n-heptane chloroform 4:1 by vortexing for 5 seconds. The sample was dropped onto a 20X 20cm WhatmanLK6D silica gel 60A TLC plate and dried in a gravity convection oven at 80 ℃ for 15 to 20 minutes. The plates were cooled to room temperature and chromatographed at room temperature in isooctane, diethyl ether, glacial acetic acid 75:25:2 for 60 minutes. The plates were dried in a gravity convection oven at 80 ℃ for 30 minutes, cooled to room temperature, and wrapped in Saran^TMIn Wrap. Plates were exposed to fluorescent imaging plates overnight, scanned on a Typhoon phosphor imager (Molecular Dynamics), and analyzed using Imagequant software (Molecular Dynamics).

Results

Treatment of primary rat hepatocytes with gemcabene significantly and concentration-dependently reduced cholesterol synthesis by 61.5 + -1.8 (SEM)% (p) at concentrations of 10 μ M and 30 μ M, respectively<1.8×10^-8) And 90.0. + -. 0.8% (p)<1.0×10^-10) (Table 7). Triglyceride synthesis was also reduced by 8.9 + -4.5% (p) dose-dependently at 10. mu.M and 30. mu.M concentrations, respectively<0.12) and 72.1. + -. 2.9% (p)<4.1×10^-8) However, only 30 μ M dose achieved statistical significance. In these cells, the HMG-CoA reductase inhibitor atorvastatin significantly reduced cholesterol synthesis by 93.7 + -0.4% (p) at a dose of 1 μ M<3.0×10^-10) Without affecting triglyceride synthesis. acetyl-CoA carboxylase (ACC) inhibitor CE 156860 did not significantly inhibit cholesterol synthesis at 3 μ M, but significantly reduced triglyceride synthesis 87.6 + -0.6% (p)<0.0031). (Table 7)

The data indicate that gemcabene is effective in inhibiting the synthesis of cholesterol and triglycerides in primary rat hepatocyte cultures. This effect of gemcabene is different from statin, atorvastatin, which inhibits cholesterol synthesis, and ACC inhibitor CE 156860, which inhibits triglyceride synthesis. The inhibitory properties of gemcabene suggest that it may affect the cholesterol and triglyceride synthetic pathways individually or the common pathway of both synthetic pathways.

TABLE 9

The% change was calculated relative to vehicle cells. Each n represents a separate experiment, which was performed in triplicate using hepatocytes isolated from each animal. P values were calculated by two-tailed test.

*(p＜1.8x10^-8)，n＝3；**(p＜1.0x10^-10)，n＝3；***(p＜3.0x10^-10)，n＝3；

(p＜4.1x10^-8)，n＝3；(p < 0.0031), n is 1; ns is not significant.

Example 6

Inhibition of plasma and hepatic cholesterol and triglyceride synthesis in C57/BL6, apoB100/lp (a) mice.

Male mice 8 to 12 weeks old were used in all studies. apoB100/lp (a) (mouse crosses for human apoB100 and human apo (a)) mice were obtained from Charles River. Eight animals per treatment group.

In this study, gemcabene was administered at 30 and/or 100 mg/kg. Simvastatin was administered at 3 mg/kg. In vehicle (1.5% carboxymethylcellulose, 0.15% Tween 20) with Polytron^TMThe balance water) and no vortexing, and is administered orally using a dose volume of 0.1mL/10g body weight.

Mice were acclimated to a reverse 12-hour light/12-hour dark cycle for a minimum of 7 days prior to administration of the first dose of test drug or vehicle. The test drugs and vehicle were administered by oral gavage approximately 2 hours prior to the mid-dark phase. Test drugs and vehicle were administered once daily. 30 minutes after the eighth dose, 12.5. mu. Ci 2¹⁴C]Sodium acetate. In [ 2 ]¹⁴C]4 hours after sodium acetate administration, mice were euthanized and bled by cardiac puncture to collect plasma. Individual plasma samples were obtained by placing whole blood samples into EDTA-loaded centrifuge tubes and centrifuging. The plasma was transferred to a new tube and stored at-20 ℃ until measurement [ 2 ]¹⁴C]Labeled cholesterol.

The liver sample was rapidly frozen in liquid nitrogen and stored at-80 ℃ until assayed¹⁴C]Labeled cholesterol and triglycerides.

Measurement of plasma [ 2 ]¹⁴C]Labeled cholesterol. Frozen plasma samples (0.2-0.4mL volume) were thawed and adjusted to a total volume of 1mL with physiological saline. Further, 0.025. mu. Ci 2¹⁴H]Cholesterol (Perkin Elmer Life sciences Inc. Boston, Massachusetts) was added to each sample and to 2 or 3 spiked controls in 7-mL scintillation vials as internal extraction standards. Freshly prepared 10% KOH solution was then added at 2.5 mL/sample. The sample was vortexed and saponified at 75 ℃ for 1 hour. After the samples were cooled, each sample was extracted with 2.5mL of petroleum ether to give μ Ci¹⁴C]Labeled cholesterol, and shaken for 10 minutes, then centrifuged at 0 ℃ for 10 minutes. The organic phase was then transferred to scintillation vials and evaporated under nitrogen at 37 ℃ head block. Each sample was then dissolved in 0.25mL of 2 parts chloroform and 1 part methanol by sonication. To each sample and spiked control was added 5mL of scintillator for direct [ 2 ] use in a Packard 2500TR Tri-Carb liquid scintillation analyzer³H]And 2¹⁴C]Degradation Per Minute (DPM) recordings. The value of [ 2 ] in each sample³H]The recovery rate of cholesterol is compared with the average total amount in the spiked control to determine [ 2 ]¹⁴C]Counts are taken as a percentage correction of variance.

Measuring liver [ 2 ]¹⁴C]Labeled cholesterol and triglycerides. During the sample weighing procedure and assay preparation, the frozen liver samples were stored on dry ice. Each sample was incubated in ice-cold methanol; homogenized in 0.5N acetic acid solution. The homogenized sample was transferred to a glass vial, and 50. mu.L of [ mu ] L in ethanol³H]Total 1x 10⁵DPM, and 15mL of chloroform was added to each sample. The sample was then vortexed and centrifuged at 3000rpm for 20 minutes at room temperature to separate the phases. The lower chloroform layer was transferred to a new vial. The remaining upper aqueous phase was re-extracted 2 times with 15mL of chloroform in a similar manner. The chloroform phases of each sample were pooled, then washed 2 times with 15mL of 0.88% KCL, vortexed for 20 seconds, and centrifuged for 20 minutes. The aqueous phase was discarded, and the chloroform phase was washed 2 times with 15mL of 1 part methanol and 1 part water in a similar manner, and the aqueous phase was discarded. The chloroform sample was evaporated under nitrogen, andresuspended in 600. mu.L chloroform and 30. mu.L removed for scintillation counting. Sample volumes were adjusted for corrected percent recovery and calculated loading volumes based on total liver weight. The calculated volume was dropped on a TLC plate and dried in an oven at 80 ℃. After the plate was cooled to room temperature, it was developed in 102mL of isooctane, diethyl ether, glacial acetic acid 75:25:2, respectively. The plates were dried in an oven at 80 ℃ and allowed to cool to room temperature, wrapped with plastic wrap, and exposed to a fluorescent imaging screen. Imagequant software was used to quantify cholesterol and triglyceride bands.

As measured in plasma, 100mg/kg of gemcabene significantly inhibited cholesterol synthesis by 60%, and 3mg/kg of simvastatin by 59% (fig. 3). The results show that 100mg/kg of gemcabene inhibits newly synthesized cholesterol 65% and triglycerides 66%, while only 3mg/kg of simvastatin reduces cholesterol synthesis by 82% in liver tissue (figure 3 and table 10).

The values shown in figure 3 are mean ± SEM, with n-8 for each group. Veh-vehicle control, Gem-100 mg/kg gemcabene, simva-3 mg/kg simvastatin. These% of + p-value based on two-tailed t-test in one-way ANOVA<0.05 based on the formula [ 2 ], [¹⁴C]Two-tailed t-test of cholesterol vs. vehicle values of p<0.05. Table 10 provides the results showing the synthesis of hepatic triglyceride and cholesterol (,) for apoB100/lp (a) by gemcabene and simvastatin in mice¹⁴C]Sodium acetate) influence.

Watch 10

Relative to "t test based on two-tailed student¹⁴C]P-value of vehicle on Cholesterol<0.05

n-8 mice/group; ns is not significant.

Reduction of liver lipids, particularly liver TG, is useful for treating or preventing NASH. In addition, gemcabene increases the oxidation of fatty acids, further reducing the tendency to develop fatty liver.

Example 7

In vivo efficacy studies of gemcabene in the STAM model of non-alcoholic steatohepatitis (NASH)

Materials and methods

Test substance: gemcabene is supplied by gemshire Therapeutics inc. To prepare the dosing solution, gemcabene was weighed according to the formulation instructions and vehicle [ purified water ]]And (4) dissolving. TelmisartanPurchased from Boehringer Ingelheim GmbH (Germany) and dissolved in pure water.

Induction of NASH

NASH was induced in 40 male rats by a single subcutaneous injection of 200 μ g streptozotocin (STZ, Sigma-Aldrich, USA) solution at 2 days postnatal and fed with a high fat diet (HFD, 57 kcal% fat, catalog No. HFD32, CLEA Japan, Japan) after 4 weeks of age.

Route of administration

Vehicle (control) was administered orally in a volume of 10 mL/kg.

Gemcabene was administered orally in a volume of 10 mL/kg.

Telmisartan is administered orally in a volume of 10 mL/kg.

The treatment dose is as follows:

gemcabene was administered once daily at doses of 30, 100 and 300 mg/kg.

Telmisartan was administered once daily at a dose of 10 mg/kg.

Animals: c57BL/6 mice (females 14 days pregnant) were obtained from Japan SLC, Inc. All animals used in the study were bred and cared for according to the Japanese Pharmacology Society Guidelines for animal use (Japanese Pharmacology Society Guidelines for animal use).

Environment: animals were maintained in SPF facilities under controlled temperature (23 + -2 deg.C), humidity (45 + -10%), lighting (12 hours artificial light and dark cycle; light from 8:00 to 20:00) and with indoor air exchange. The laboratory is pressurized to prevent contamination of the facility.

Feeding animals: animals were housed in TPX cages (CLEA Japan) with a maximum of 4 mice per cage. Sterile Paper-clean (Japan SLC) was used for the pads and replaced once a week.

Food and water: sterile solid High Fat Diet (HFD) was provided ad libitum and placed in a metal lid on top of the cage. Purified water was supplied ad libitum from a water bottle fitted with a rubber stopper and straw. The water bottles were replaced weekly and cleaned and sterilized in an autoclave and reused.

Identification of animals and cages: mice were identified by ear holes. Each cage is marked with a specific identification code.

Whole blood and plasma biochemical measurements: an 8 hour fasting blood sample was taken from the facial vein 3 days prior to termination.

An 8-hour fasting blood glucose was measured in whole blood using Life Check (EIDIA co. ltd., Japan). For plasma biochemistry, 8 hour fasting blood was collected in polypropylene tubes with anticoagulant (Novo-Heparin, mochida pharmaceutical, Japan) and centrifuged at 1,000xg for 15 minutes at 4 ℃. The supernatant was collected and stored at-80 ℃ until use. Plasma insulin levels were quantified by an ultrasensitive mouse insulin ELISA kit (Ultra Sensitive mouse insulin ELISA kit, Morinaga Institute of Biological Science, Inc., Japan).

On the day of termination, non-fasting blood glucose was measured in whole blood using Life Check. For plasma biochemistry, non-fasting blood was collected in polypropylene tubes with anticoagulant (Novo-Heparin) and at 4 ℃ CCentrifuge at 1,000Xg for 15 minutes. The supernatant was collected and stored at-80 ℃ until use. Plasma ALT, AST, ALP, GGT, BUN, creatinine, and total bilirubin levels were measured by FUJI DRI-CHEM 7000(Fujifilm Corporation, Japan). By EnzyChrom^TMKetone body assay kits (BioAssay Systems, USA) quantify plasma ketone body levels.

Liver biochemical measurements

Measurement of hepatic triglyceride content: a total lipid extract from the liver was obtained by the Folch method (Folch J. et al, J.biol. chem.1957; 226: 497). Liver samples were homogenized in chloroform-methanol (2:1, v/v) and incubated overnight at room temperature. After washing with chloroform-methanol-water (8:4:3, v/v/v), the extract was evaporated to dryness and dissolved in isopropanol. The hepatic triglyceride content was measured by the triglyceride E test (Wako Pure Chemical Industries).

Measurement of hepatic hydroxyproline content: to quantify the hepatic hydroxyproline content, frozen liver samples were treated by the following alkaline-acidic hydrolysis method. Liver samples were defatted with 100% acetone, dried in air, dissolved in 2N NaOH at 65 ℃ and autoclaved at 121 ℃ for 20 minutes. The lysed sample (400. mu.L) was acid hydrolyzed with 400. mu.L of 6N HCl at 121 ℃ for 20 minutes and neutralized with 400. mu.L of 4N NaOH containing 10mg/mL of activated carbon. To the sample was added AC buffer (2.2M acetic acid/0.48M citric acid, 400. mu.L), followed by centrifugation to collect the supernatant. A standard curve of hydroxyproline was constructed using serial dilutions of trans-4-hydroxy-L-proline (Sigma-Aldrich) starting at 16. mu.g/mL. The prepared samples and standards (400. mu.L each) were mixed with 400. mu.L of chloramine T solution (Wako Pure Chemical Industries) and incubated for 25 minutes at room temperature. The sample was then mixed with an Ehrlich solution (400 μ L) and heated at 65 ℃ for 20 minutes to develop the color. After the samples were cooled on ice and centrifuged to remove the precipitate, the optical density of each supernatant was measured at 560 nm. The hydroxyproline concentration was calculated from a standard curve of hydroxyproline. Protein concentrations of liver samples were determined using the BCA protein assay kit (Thermo FisherScientific, USA) and used to normalize the calculated hydroxyproline values. Hepatic hydroxyproline levels are expressed in μ g/mg protein.

Histological analysis: for HE staining, sections were cut from pre-fixed paraffin blocks of liver tissue in buuin's solution and stained with Lillie-Mayer's hematoxylin (Muto Pure Chemicals co., ltd., Japan) and eosin solution (Wako Pure Chemical Industries). The NAFLD Activity Score (NAS) was calculated according to the Kleiner criteria (KleinerDE et al, Hepatology, 2005; 41: 1313). To observe collagen deposition, buan's fixed liver sections were stained using picric acid sirius red solution (Waldeck, Germany).

For quantitative analysis of fibrotic regions, bright field images of sirius red stained sections were captured around the central vein using a 200-fold magnified digital camera (DFC 295; Leica, Germany), and positive regions were measured at 5 fields per section using ImageJ software (National Institute of Health, USA).

Statistical analysis was performed on GraphPad Prism 6(GraphPad Software inc., USA) using Bonferroni multiple comparison tests. P values <0.05 were referred to as statistically significant. Results are expressed as mean ± SD.

Experimental design and treatment

Research group

Group 1: normal medium

Eight normal mice (not administered streptozotocin) were orally administered a vehicle in a volume of 10mL/kg once daily from 6 to 9 weeks of age [ purified water ].

Group 2: vectors in the streptomycin-induced NASH model

Eight NASH mice were orally administered with vehicle in a volume of 10mL/kg once a day from 6 to 9 weeks of age.

Group 3: gemcarbin 30mg/kg in streptomycin-induced NASH model

Eight NASH mice were orally administered vehicle supplemented with gemcabene at a dose of 30mg/kg once a day from 6 to 9 weeks of age.

Group 4: gemcarbin 100mg/kg in streptomycin-induced NASH model

Eight NASH mice were orally administered vehicle supplemented with gemcabene at a dose of 100mg/kg once a day from 6 to 9 weeks of age.

Group 5: 300mg/kg Gemcarbin streptozotocin induced NASH model

Eight NASH mice were orally administered with gemcabene-supplemented vehicle at a dose of 300mg/kg once daily from 6 to 9 weeks of age.

Group 6: telmisartan 10mg/kg in streptozotocin-induced NASH model

Eight NASH mice were orally administered pure water supplemented with telmisartan at a dose of 10mg/kg once a day from 6 to 9 weeks of age.

Table 11 below summarizes the treatment schedule:

TABLE 11

Animal monitoring and sacrifice

Viability, clinical signs and behavior were monitored daily. Body weights were recorded before treatment. Mice were observed for significant clinical signs of toxicity, moribundity and death approximately 60 minutes after each administration. Animals were sacrificed at 9 weeks of age by direct cardiac puncture exsanguination under isoflurane anesthesia (Pfizer Inc.).

Results

Weight change and general condition

The mean body weight of the vehicle group in NASH was significantly lower than the body weight of the vehicle group in normal during the treatment period. The mean body weight of the telmisartan group was significantly lower than that of the vehicle group in NASH on days 10 to 21. Mean body weight did not change significantly between the vehicle and gemcabene treated groups in NASH during the treatment period (figure 4). During the treatment period, one mouse in the telmisartan group was found to die before reaching day 21.

Body weight on termination day

The vehicle group in NASH showed a significant reduction in mean body weight on the day of termination compared to the normal vehicle group. The telmisartan group on the day of termination showed a significant reduction in mean body weight compared to the vehicle group in NASH. There was no significant difference in mean body weight on the day of termination between the vehicle group and the gemcabene treated group in NASH (figure 5A and table 13).

Liver weight and liver to body weight ratio

The vehicle group in NASH showed a significant increase in mean liver weight compared to the vehicle group in normal. The gemcabene 100 and 300mg/kg groups showed a significant increase in mean liver weight compared to the vehicle group in NASH. The telmisartan group showed a significant reduction in mean liver weight compared to the vehicle group in NASH. There was no significant difference in mean liver weight between the vehicle group and the gemcabene 30mg/kg group in NASH (figure 5B and table 13).

The vehicle group in NASH showed a significant increase in mean liver to body weight ratio compared to the vehicle group in normal. The gemcabene 100 and 300mg/kg groups showed a significant increase in mean liver to body weight ratio compared to the vehicle group in NASH. There was no significant difference in mean liver to body weight ratio between the vehicle group and any other treatment group in NASH (fig. 5C and table 12).

Table 12: body weight and liver weight

Biochemistry

After 8 hours of fasting for 3 days before termination

Fasting whole blood glucose: the vehicle group in NASH showed a significant increase in fasting whole blood glucose levels compared to the vehicle group in normal. The telmisartan group showed a significant increase in fasting whole blood glucose levels compared to the vehicle group in NASH. There was no significant difference in fasting whole blood glucose levels between the vehicle group and the gemcabene treated group in NASH (fig. 6A and table 13).

Fasting plasma insulin: the vehicle group in NASH showed a significant reduction in fasting plasma insulin levels compared to the vehicle group in normal. There was no significant difference in fasting plasma insulin levels between the vehicle group and any other treatment group in NASH (fig. 6B and table 13).

At the time of termination

Whole blood glucose: the vehicle group in NASH showed a significant increase in whole blood glucose levels compared to the vehicle group in normal. The telmisartan group showed a significant increase in whole blood glucose levels compared to the vehicle group in NASH. There was no significant difference in whole blood glucose levels between the vehicle group and the gemcabene treated group in NASH (fig. 7A and table 13).

Plasma ALT: the vehicle group in NASH showed significantly increased plasma ALT levels compared to the vehicle group in normal. The gemcabene 100mg/kg group showed a significant reduction in plasma ALT levels compared to the vehicle group in NASH. There was no significant difference in plasma ALT levels between the vehicle group and any other treatment group in NASH (fig. 7B and table 13).

Plasma AST: there was no significant difference in plasma AST levels between the vehicle group and any treatment group in NASH (fig. 7C and table 13).

Plasma ALP: the gemcabene 100 and 300mg/kg groups and the telmisartan group showed significantly increased plasma ALP levels compared to the vehicle group in NASH. There was no significant difference in plasma ALP levels between the vehicle group and any other treatment group in NASH (fig. 7D and table 13).

Plasma GGT: there was no significant difference in plasma GGT levels between the vehicle group and any treatment group in NASH (fig. 7E and table 13).

Plasma BUN: the telmisartan group showed a significant increase in plasma BUN levels compared to the vehicle group in NASH. There was no significant difference in plasma BUN levels between the vehicle group and any other treatment group in NASH (fig. 7F and table 13).

Plasma creatinine: the vehicle group in NASH showed a significant reduction in plasma creatinine levels compared to the vehicle group in normal. The gemcabene 300mg/kg group showed a significant increase in plasma creatinine levels compared to the vehicle group in NASH. There was no significant difference in plasma creatinine levels between the vehicle group and any other treatment group in NASH (fig. 7G and table 13).

Plasma total bilirubin: there was no significant difference in plasma total bilirubin levels between the vehicle group and any of the treatment groups in NASH (fig. 7H and table 13).

Plasma ketone bodies: the vehicle group in NASH showed a significant increase in plasma ketone body levels compared to the vehicle group in normal. There was no significant difference in plasma ketone body levels between the vehicle group and any other treatment group in NASH (fig. 7I and table 13).

Hepatic triglycerides: the vehicle group in NASH showed a significant increase in hepatic triglyceride content compared to the vehicle group in normal. The telmisartan group showed a significant reduction in hepatic triglyceride content compared to the vehicle group in NASH. There was no significant difference in hepatic triglyceride content between the vehicle group and the gemcabene treated group in NASH (fig. 7J and table 13).

Hepatic hydroxyproline: there was no significant difference in hepatic hydroxyproline content between the vehicle group and any treatment group in NASH (fig. 7K and table 13).

Table 13: biochemistry

After fasting for 8 hours 3 days before termination (day 18)

At the end of the day (day 21)

Histological analysis

HE staining and NAFLD activity scoring

NASH is defined by the presence and pattern of specific histological abnormalities in liver biopsies. NAFLD Activity Score (NAS) is a composite score developed as a tool to measure NAFLD changes during therapeutic trials. NAS is a composite score consisting of three components, including steatosis, lobular inflammation and hepatocellular ballooning (table 14). The NAS score was defined as the unweighted sum of the scores for steatosis, lobular inflammation and hepatocellular ballooning. The steatosis grade was quantified as the percentage of hepatocytes containing fat droplets. The sirius red staining intensity of collagen in the peripheral area of the center of the hepatic lobule is evaluated histologically, and the stage of hepatic fibrosis is evaluated separately from NAS.

Liver sections from the vehicle group in NASH showed microbubble and bullous fatty deposits, hepatocyte ballooning and inflammatory cell infiltration compared to the vehicle group in normal. The vehicle group in NASH showed a significant increase in NAS compared to the vehicle group in normal. The gemcabene 30 and 300mg/kg groups and the telmisartan group showed a significant reduction in NAS compared to the vehicle group in NASH (fig. 8 and 9A-9C and table 14).

Table 14: NAFLD activity score

Hepatic fibrosis

Dyeing with sirius red

Sirius red stained liver sections were evaluated to determine liver fibrosis. Liver sections from the vehicle group in NASH showed increased collagen deposition in the pericentral region of the liver lobule compared to the vehicle group in normal. All groups showed a significant reduction in fibrotic area compared to the vehicle group in NASH (figure 10 and table 15).

Table 15: histological analysis

Summary and discussion

Telmisartan has been demonstrated to have anti-steatosis, anti-inflammatory and anti-fibrotic effects in the STAM mice and was therefore used as a positive control in this study. Consistent with the historical data of SMC Laboratories, telmisartan treatment significantly reduced hepatic triglyceride content, NAS and fibrotic area.

Gemcabene significantly reduced the area of fibrosis compared to the vehicle group in NASH, indicating an anti-fibrotic effect in this study. Medium and high doses of gemcabene increased plasma ALP levels compared to vehicle group in NASH. High doses of gemcabene also increased plasma creatinine levels compared to the vehicle group in NASH. Plasma ALT levels were reduced in the gemcabene treated group and were statistically significant in the intermediate dose gemcabene group. The low and high dose gemcabene reduced NAS compared to the vehicle group in NASH. In NAS, high doses of gemcabene reduced steatosis and ballooning scores to an extent comparable to telmisartan. Since hepatocyte ballooning is thought to arise from oxidative stress-induced hepatocyte injury and is associated with disease progression in NASH (Fujii H et al j. atheroscler.thromb.2009; 16:893, Rangwala F et al j. pathol.2011; 224:401), it suggests that gemcabene ameliorates NASH pathology by inhibiting hepatocyte injury and ballooning-like attenuation cell formation. In summary, in this study gemcabene showed anti-fibrotic effects at all doses tested and demonstrated anti-NASH and liver-protective effects on liver pathology in tam mice. These data indicate that gemcabene leads to liver improvement and may positively affect inflammatory and/or metabolic-related molecules, which can be assessed for specific targets by liver gene expression analysis or immunohistochemistry

Example 8

Hepatic lipids in male Sprague-Dawley rats treated with gemfibrozil or gemcabene.

Fifty-six male Sprague-Dawley rats were obtained from Charles River Laboratories. All animals were allowed free intake of normal rat food (Ralston-Purina) and drinking water in a temperature controlled room under a 12 hour light, 12 hour dark cycle starting at 6 a.m. illumination. Rats were assigned to 7 groups of 8 rats per group. Rats were dosed daily at 6 to 10 am by oral gavage using a suspension vehicle of 1.5% carboxymethylcellulose plus 0.2% Tween-20 (vehicle). Control animals received vehicle alone. The vehicle volume administered was 0.25% of body weight. PD 72953 is gemcabene. CI-719 is gemfibrozil. The compounds were administered daily to seven treatment groups for fourteen consecutive days as shown in table 16.

TABLE 16

Group of	Medicine	Dosage form
			1	Control	-----
2	Gemfibrozil	100 mg/kg/day
			3	PD 72953	1 mg/kg/day
4	PD 72953	3 mg/kg/day
			5	PD 72953	10 mg/kg/day
6	PD 72953	30 mg/kg/day
			7	PD 72953	100 mg/kg/day

Animals were sacrificed on the last day and liver lipids were extracted and triglyceride and cholesterol levels were determined by the method of Homan and Anderson, Journal of chromatography B,708(1998), 21-26. Approximately 500mg of liver fragments were extracted for lipids and liver proteins were determined.

For rats treated with vehicle (control) or the indicated dose of gemfibrozil or gemcabene, the data are shown as mean ± SEM of μ g hepatic triglyceride/mg hepatic protein (fig. 11A) or μ g hepatic unesterified cholesterol/mg hepatic protein (fig. 11B). Statistical analysis was ANOVA with post hoc Fisher PLSD (. p <0.05,. p < 0.01).

Example 9

Fibrinogen levels in rats

Plasma was collected from the rats in example 8 after sacrifice. Plasma fibrinogen plasma levels were determined by electroimmunoassay. Gemfibrozil at 100 mg/kg/day and PD 72953 at 30 mg/kg/day and 100 mg/kg/day showed significant reductions in plasma fibrinogen levels of 31, 52, and 57%, respectively, as compared to the control. Data are shown in figure 12 as mean ± SEM of percent control fibrinogen levels. P <0.001, double-sided unpaired t-test, compared to control.

Example 10

Fibrinogen levels in humans

In a post hoc analysis of fibrinogen for an 8 week, double-blind, randomized, placebo-controlled, dose-ranging study (study a4141001) of efficacy and safety of gemcabene (CI-1027) treatment of hypercholesterolemic patients administered as monotherapy or in combination with atorvastatin. Blood samples were collected at the beginning (visit T5; week 0) and end (visit T8, week 8) to measure fibrinogen.

The analysis of covariance (ANCOVA) method used in original study a4141001 was again used for these analyses. For fibrinogen, baseline was defined as the last pre-treatment follow-up, and endpoints were the last follow-up to and including the second day after the last dose was administered. The least squares mean and p-value of fibrinogen changes from baseline were calculated using an ANCOVA model with baseline lipid values and effects of treatment. In addition, median and p-value for percent change from baseline.

All patients enrolled in the fibrinogen assay were randomized and received at least 1 dose of study drug. In addition, these patients must have one baseline and at least 1 valuable post-baseline measurement. Thus, this is a definition of a modified intent-to-treat (MITT) population.

Gemcabene 300, 600 and 900mg monotherapy increased fibrinogen from baseline to endpoint by an average of 24.2, 23.6 and 12.9, respectively, compared to 37.2 in the placebo group. As shown in table 17, the data for rank switching did not show significant differences between gemcabene and placebo.

TABLE 17

Change from baseline to endpoint of gemcabene monotherapy relative to placebo-fibrinogen

(modified treatment intent)

Pbo is placebo; difference gemxxmg-placebo; SE is the standard error; CI-confidence interval

As shown in table 18, co-administration of 600mg of gemcabene with atorvastatin aggregated over the dose range showed a reduction in fibrinogen over atorvastatin monotherapy-31.6 (p ═ 0.0177). A smaller decrease was observed when 300 and 900mg of gemcabene was co-administered with atorvastatin.

Watch 18

Change from baseline to endpoint of gemcabene + atorvastatin relative to atorvastatin-fibrinogen

(modified treatment intent)

Gemc + Ator ═ gemcabene combination atorvastatin; ator ═ atorvastatin monotherapy; SE is the standard error;

CI is confidence interval; the difference is (Gem + Ator) -Ator.

The normal range for fibrinogen is about 150-300 mg/dL. To observe the effect of either gemecar monotherapy or the gemcabene combination atorvastatin in subjects with fibrate levels above the normal fibrinogen range, data for a subset of subjects with baseline fibrinogen levels > 400mg/dL were examined. Table 19 shows the effect of different doses of gemcabene alone, atorvastatin alone or various doses of a combination of gemcabene and atorvastatin on changes in baseline fibrinogen levels. Statin combination treatment of 600mg gemcabene with 10, 40 or 80mg atorvastatin resulted in a reduction of 22.5, 10.8 and 16.8%, respectively.

Watch 19

Table 20 shows data for gemcabene at each dose and atorvastatin at any dose. 600mg of gemcabene in combination with atorvastatin showed a 17.1% reduction.

Watch 20

Table 21 shows data for 300, 600, and 900mg gemcabene (Gem) monotherapy.

TABLE 21

Gem is gemcabene, SE is standard error, CI is confidence interval, and the difference is Gem-placebo.

Table 22 shows data for the fused atorvastatin (Ator.) doses and gemcabene (gem) for each dose for placebo. The reduction in gemcabene in combination with atorvastatin showed a significant reduction of 91.7mg/dL (p ═ 0.0002) compared to atorvastatin alone.

TABLE 22

Gem + Ator is gemcabene combined atorvastatin; ator is atorvastatin monotherapy; SE is SE standard error, CI is confidence interval, and the difference is (Gem + Ator) -atom.

Example 11

Representative examples of fixed dose combinations are provided in table 16.

TABLE 16

All publications and patents cited in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. If the meaning of a term in any patent or publication incorporated by reference conflicts with the meaning of the term used in the present disclosure, the meaning of the term in the present disclosure shall govern. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments of the present invention. One skilled in the art will readily recognize from such discussion, and from the accompanying drawings and claims, that various changes, modifications and variations can be made therein without departing from the spirit and scope of the invention as defined in the following claims.

Claims

1. A method of treating a subject with type IIb hyperlipidemia comprising administering to the subject gemcabene in combination with a low or medium dose of a statin.

2. The method of claim 1, wherein the daily dose of gemcabene administered is from about 50mg to about 900mg and the daily dose of statin administered is from about 1mg to about 60 mg.

3. The method of claim 1 or claim 2, wherein the daily dose of gemcabene is from about 50mg to about 900mg per day, wherein:

a. the statin is atorvastatin and the daily dose of atorvastatin is from about 10mg to about 60 mg;

b. the statin is rosuvastatin and the daily dose of rosuvastatin is from about 5mg to about 30 mg;

c. the statin is simvastatin, and the daily dose of simvastatin is from about 5mg to about 60 mg;

d. the statin is pravastatin and the daily dose of pravastatin is from about 10mg to about 60 mg;

e. the statin is lovastatin and the daily dose of lovastatin is from about 20mg to about 60 mg;

f. the statin is fluvastatin and the daily dose of fluvastatin is from about 20mg to about 40 mg; or

4. The method of claim 3, wherein the daily dose of gemcabene is from about 150mg to about 600 mg.

5. The method of claim 3, wherein the daily dose of gemcabene is 150mg, 300mg, 450mg, 600mg, or 900 mg.

6. The method of any one of claims 3-5, wherein the daily dose of atorvastatin is from about 10mg to about 40 mg.

7. The method of any one of claims 3-5, wherein the daily dose of rosuvastatin is from about 10 to about 20 mg.

8. The method of any one of claims 3-5, wherein the daily dose of simvastatin is from about 10mg to about 20 mg.

9. The method of any one of claims 3-5, wherein the daily dose of pravastatin is from about 10mg to about 40 mg.

10. The method of any of claims 3-5, wherein the daily dose of lovastatin is about 20 to about 40 mg.

11. The method of any one of claims 3-5, wherein the daily dose of fluvastatin is about 40 mg.

12. The method of any one of claims 3-5, wherein the daily dose of pitavastatin is from about 1mg to about 3 mg.

13. The method of any one of claims 1-12, wherein the subject has one or more of: familial complicated with hyperlipidemia, metabolic syndrome, impaired glucose tolerance, inflammatory disorders, obesity, NASH, NAFLD, alcoholic liver disease, or primary biliary cirrhosis.

14. The method of any one of claims 3-13, wherein the statin and gemcabene are administered in a fixed dose combination.

15. The method of any one of claims 1-14, wherein the subject's plasma triglyceride level is reduced to less than 150mg/dl within 8 weeks of administration of gemcabene and the statin.

16. The method of any one of claims 1-15, wherein the subject's plasma LDL cholesterol level is reduced to less than 130mg/dl within 8 weeks of administration of gemcabene and the statin.

17. The method of any one of claims 1-15, wherein the subject's ApoB level is reduced to less than 120mg/dl within 8 weeks of administration of gemcabene and the statin.

18. The method of any one of claims 1-17, wherein the subject's risk of myopathy is not increased relative to the risk of administration of the statin alone.

19. The method of any one of claims 1-17, wherein the subject's risk of myositis is not increased relative to the risk of administration of the statin alone.

20. The method of any one of claims 1-17, wherein the subject's risk of rhabdomyolysis is not increased as compared to the risk of administration of the statin alone.

21. The method of any one of claims 1-20, wherein the subject is administered an additional lipid lowering agent, a PCSK9 inhibitor, a cholesterol absorption inhibitor, an ACC inhibitor, an ApoC-III inhibitor, an ACL inhibitor, a prescription fish oil, or a CETP inhibitor.

22. The method according to claim 21, wherein the additional lipid lowering agent is ezetimibe.

23. The method of any one of claims 1-22, wherein the subject is at reduced risk of having a primary cardiovascular event.

24. The method of any one of claims 1-23, wherein the subject is at reduced risk of having a secondary cardiovascular event.

25. A fixed dose combination comprising gemcabene in an amount from about 50mg to about 900mg and a statin in an amount from about 1mg to about 60 mg.

26. The fixed-dose combination of claim 25, wherein the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin or pitavastatin, wherein:

a. the amount of atorvastatin is from about 10mg to about 60 mg;

b. the amount of rosuvastatin is from about 5mg to about 30 mg;

c. simvastatin in an amount from about 10mg to about 60 mg;

d. the amount of pravastatin is from about 10mg to about 60 mg;

e. the amount of lovastatin is about 20mg to about 40 mg;

f. the amount of fluvastatin is from about 20mg to about 60 mg; or

g. The amount of pitavastatin is from about 1mg to about 4 mg.

27. The fixed dose combination of claim 26, wherein the amount of gemcabene is about 150mg to about 600 mg.

28. The fixed dose combination of claim 27, wherein the amount of gemcabene is from about 150 to about 300.

29. The fixed dose combination of claim 27, wherein the amount of gemcabene is about 300mg to about 450 mg.

30. The fixed dose combination of claim 26, wherein the amount of gemcabene is 150mg, 300mg, 450mg, 600 mg.

31. The fixed dose combination of claim 30, wherein

h. The amount of atorvastatin is 10mg, 20mg or 40 mg;

i. the amount of rosuvastatin is 10mg or 20 mg;

j. the amount of simvastatin is 10mg or 20 mg;

k. the amount of pravastatin is 10mg or 20 mg;

the amount of said lovastatin is 20mg or 40 mg;

the amount of fluvastatin is 20mg or 40 mg; or

n. the amount of said pitavastatin is 1mg, 2mg or 3 mg.

32. The fixed dose combination of claim 30, wherein the fixed dose combination comprises 150mg gemcabene and 10mg atorvastatin.

33. The fixed dose combination of claim 30, wherein the fixed dose combination comprises 150mg gemcabene and 20mg atorvastatin.

34. The fixed dose combination of claim 30, wherein the fixed dose combination comprises 150mg gemcabene and 40mg atorvastatin.

35. The fixed dose combination of claim 30, wherein the fixed dose combination comprises 300mg gemcabene and 10mg atorvastatin.

36. The fixed dose combination of claim 30, wherein the fixed dose combination comprises 300mg gemcabene and 20mg atorvastatin.

37. The fixed dose combination of claim 30, wherein the fixed dose combination comprises 300mg gemcabene and 40mg atorvastatin.

38. The fixed dose combination of claim 30, wherein the fixed dose combination comprises 600mg gemcabene and 10mg atorvastatin.

39. The fixed dose combination of claim 30, wherein the fixed dose combination comprises 600mg gemcabene and 20mg atorvastatin.

40. The fixed dose combination of claim 30, wherein the fixed dose combination comprises 600mg gemcabene and 40mg atorvastatin.

41. The fixed dose combination of claim 30, wherein the fixed dose combination comprises 900mg of gemcabene and 10mg of atorvastatin.

42. The fixed dose combination of claim 30, wherein the fixed dose combination comprises 900mg gemcabene and 20mg atorvastatin.

43. The fixed dose combination of claim 30, wherein the fixed dose combination comprises 900mg gemcabene and 40mg atorvastatin.

44. A method for treating or preventing hepatic steatosis, comprising administering to a subject in need thereof an effective amount of a compound of formula (I):

or a pharmaceutically acceptable salt, hydrate, solvate, or mixture thereof, wherein:

(a) each occurrence of m is independently an integer ranging from 0 to 5;

(b) each occurrence of n is independently an integer in the range of 3 to 7;

Wherein:

45. A method for treating or preventing hepatic steatosis, comprising administering to a subject in need thereof an effective amount of a compound of formula (II):

(a) each occurrence of m is independently an integer ranging from 0 to 5;

(b) each occurrence of n is independently an integer in the range of 3 to 7;

(c) x is- (CH)₂)-、–O-、–CH(OH)-、CH(CH₂OH) -, -NH-or-S-, wherein z is an integer from 0 to 4;

Wherein:

46. The method of claim 45, wherein the compound has the structure:

or a pharmaceutically acceptable salt of any of the above.

47. A method of reducing liver fat accumulation in a subject at risk for liver fat accumulation comprising administering gemcabene to the subject.

48. The method of claim 47, wherein the subject has hepatic steatosis.

49. The method of claim 47 or claim 48, wherein the subject has type IIb hyperlipidemia or familial combined hyperlipidemia.

50. The method of any one of claims 47-49, wherein the daily dose of gemcabene administered is from about 50mg to about 900 mg.

51. The method of claim 50, wherein the daily dose of gemcabene is from about 150mg to about 600 mg.

52. The method of claim 51, wherein the daily dose of gemcabene is 150mg, 300mg, 450 or 600 mg.

53. The method of any one of claims 50-52, wherein gemcabene is administered in combination with a statin.

54. The method of claim 53, wherein the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin or pitavastatin.

55. The method of any one of claims 50-54, wherein the subject is at reduced risk of developing a liver disease.

56. The method of any one of claims 49-53, wherein the subject has a liver disease.

57. The method of claim 55 or 56, wherein the liver disease is non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD) or alcoholic steatosis.

58. The method of any one of claims 47-57, wherein the subject is administered an additional cholesterol lowering agent.

59. The method of claim 58, wherein the additional lipid lowering agent is a cholesterol absorption inhibitor, a PCSK9 inhibitor, an ACC inhibitor, an ApoC-III inhibitor, an ACL-inhibitor, a prescription fish oil, or a CETP inhibitor.

60. The method of claim 59, wherein the cholesterol lowering agent is a cholesterol absorption inhibitor and the cholesterol absorption inhibitor is ezetimibe.

61. The method of claim 60, wherein the hepatic steatosis is NAFLD or NASH.

62. A method of stabilizing or reducing NAFDL Activity Score (NAS) in a subject comprising administering gemcabene to the subject.

63. The method of claim 62, wherein the daily dose of gemcabene administered is from about 50mg to about 900 mg.

64. The method of claim 63, wherein the daily dose of gemcabene is from about 150mg to about 600 mg.

65. The method of claim 64, wherein the daily dose of gemcabene is 150mg, 300mg, 450 or 600 mg.

66. The method of any one of claims 62-65, wherein gemcabene is administered in combination with a statin.

67. The method of claim 66, wherein the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin or pitavastatin.

68. The method of any one of claims 62-67, wherein the method comprises slowing, stabilizing, or reducing the progression of the steatosis component of the NAS.

69. The method of any one of claims 62 to 67, wherein the method comprises slowing progression, stabilizing or reducing the lobular inflammatory component of NAS.

70. The method of any one of claims 62-67, wherein the method comprises slowing progression of, stabilizing, or reducing the hepatocyte ballooning component of NAS.

71. The method of any one of claims 62-70, wherein the difference in NAS is no less than 1.5 points after 6 months of treatment with gemcabene.

72. A method of reducing liver fibrosis in a subject in need thereof, comprising administering gemcabene to the subject.

73. The method of claim 72, wherein the subject has NASH.

74. The method of claim 72, wherein the subject has primary biliary cirrhosis.

75. The method of claim 74, wherein the daily dose of gemcabene administered is from about 50mg to about 900 mg.

76. The method of claim 75, wherein the daily dose of gemcabene is from about 150mg to about 600 mg.

77. The method of claim 76, wherein the daily dose of gemcabene is 150mg, 300mg, 450 or 600 mg.

78. The method of any one of claims 72-77, wherein gemcabene is administered in combination with a statin.

79. The method of claim 78, wherein the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin or pitavastatin.

80. A method for reducing plasma fibrinogen levels in a subject in need thereof comprising administering gemcabene to the subject.

81. The method of claim 80, wherein the subject has a fibrinogen level greater than 300mg/dL, comprising administering gemcabene to the subject.

82. A method according to claim 81, wherein the subject has a fibrinogen level greater than 400 mg/dL.

83. The method of any one of claims 79-81, wherein the daily dose of gemcabene is from 50mg to 900 mg.

84. The method of any one of claims 80-83, wherein gemcabene is administered in combination with a statin.

85. The method of claim 84, wherein the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin or pitavastatin.

86. The method of claim 85, wherein the daily dose of the statin is 1mg to 80 mg.

87. The method of claim 86, wherein the daily dose of gemcabene is 300mg, 600mg, or 900mg and the statin is atorvastatin administered at a daily dose of 10mg, 40mg, or 80 mg.

88. The method of claim 87, wherein the daily dose of gemcabene is 600mg and the daily dose of atorvastatin is 10 mg.