WO2023026130A1

WO2023026130A1 - Pharmaceutical combination of ppar agonist(s) and sterol absorption inhibitor(s) and use thereof

Info

Publication number: WO2023026130A1
Application number: PCT/IB2022/057537
Authority: WO
Inventors: Rakesh Iswar PATIL; Srinivasa Reddy Bapuram; Sazid ALI; Bhavishya VASHIST; Santosh Kumar Rai; Anil Kumar
Original assignee: Mankind Pharma Ltd.
Priority date: 2021-08-25
Filing date: 2022-08-12
Publication date: 2023-03-02
Also published as: AR126844A1; CN117858697A; TW202313019A

Abstract

The present invention relates to pharmaceutical combinations of peroxisome proliferator-activated receptor (PPAR) agonist(s) with sterol absorption inhibitor(s), as well as to the use of these combinations for treating and/or preventing non-alcoholic fatty liver disease (NAFLD) and/or non-alcoholic steatohepatitis (NASH) and other related disorders.

Description

PHARMACEUTICAL COMBINATION OF PPAR AGONIST(S) AND STEROL ABSORPTION INHIBITOR(S) AND USE THEREOF

FIELD OF THE INVENTION

The present invention relates to pharmaceutical combination of peroxisome proliferator-activated receptor (PPAR) agonist(s) with sterol absorption inhibitor(s), as well as to the use of these combinations for treating and/or preventing non-alcoholic fatty liver disease (NAFLD) and/or non-alcoholic steatohepatitis (NASH) and other related disorders.

BACKGROUND OF THE INVENTION

Non-alcoholic fatty liver disease (NAFLD) is a major cause of chronic liver disease and is associated with significant morbidity and mortality worldwide, with a global prevalence of 25%. High incidence of NAFLD is observed in Western countries and non-Westem countries that have adopted a Western diet. NAFLD is commonly associated with components of the metabolic syndrome, type 2 diabetes mellitus and cardiovascular disease, suggesting a common mechanistic basis.

NAFLD is defined as the accumulation of excess triglyceride droplets in the liver (>5% of hepatocytes with droplets detected histologically or >5% proton density fat fraction by magnetic resonance imaging [MRI]) in people who consume little or no alcohol. NASH (non-alcoholic steatohepatitis) is a subset of NAFLD characterized by biopsy evidence of hepatocellular injury and death, inflammation and varying degrees of fibrosis. Constant worsening of this disease may ultimately lead to irreversible liver damage including fibrosis, cirrhosis and hepatocellular carcinoma.

An inability to metabolically handle free fatty acid overload-metabolic inflexibility-constitutes a core node for NAFLD pathogenesis, with resulting lipotoxicity, mitochondrial dysfunction and cellular stress leading to inflammation, apoptosis and fibrogenesis. These responses can lead to the histological phenotype of non-alcoholic steatohepatitis (NASH) with varying degrees of fibrosis, which can progress to cirrhosis. Currently, there is no established treatment for this disease.

Mol Metab. 2020 Jul 13; 101049, discloses that the insulin sentitizers such as pioglitazone along with hepatoprotective agents such as vitamin E have shown benefits for NASH histological features and are the only recommended agents in current clinical guidelines.

WO 2017/162211 discloses pharmaceutical composition for treating NAFLD, diabetes or cardiovascular diseases, as well as preventing and/or treating hepatitis, liver fibrosis, liver cirrhosis, liver cancer etc., wherein said composition comprises two components. The first component comprises famesoid X receptor agonist is selected from cholic acid (CA), lithocholic acid (LCA), obeticholic acid (OCA), chenodeoxycholic acid (CDCA), Ursodeoxycholic acid (UDCA), deoxycholic acid (DCA), at least one of GW4064, WAY-362450, PX-102 and PX20350. The second component comprises the NPC1L1 receptor inhibitor is a cholesterol absorption inhibitor compound such as ezetimibe.

IN 236792 discloses pharmaceutical composition comprising PPAR agonist(s) and sterol absorption inhibitor(s) wherein said PPAR agonist(s) are selected from gemfibrozil, clifibrate and/or fenofibrate; and said sterol absorption inhibitors are selected from statins such as lovastatin, pravastatin and/or simavastatin for the treatment of vascular condition, diabetes, obesity etc.

Despite the significant disease burden of NASH worldwide, there are currently no approved pharmacological treatments, though multiple drug candidates are in clinical development. Current interventions largely focus on lifestyle modification — healthy eating habits, weight loss and increased physical activity — to help manage the patient's metabolic syndrome.

Obesity and metabolic dysfunctions such as insulin resistance or dyslipidemia are the best-known mechanisms leading to excessive accumulation of triglycerides in hepatocytes. It is known that obesity, type 2 diabetes mellitus (T2DM), and dyslipidemia are the most common metabolic risk factors associated with both the development and progression of NAFLD. The risk of NAFLD and NASH was from 4.1- to 14-fold higher in patients with a higher BMI. Additionally, it was approximately 50% higher in men and doubled in those with diabetes. In line with these data, a recent study with 3202 individuals reported that higher BMI (overweight/obesity) is an independent, dose-dependent risk factor for fatty liver disease.

An important approach suggested by clinicians for the management of NASH is the use of combination therapies which can engage the extra-hepatic organs such as adipose tissue and the intestine. The pathogenesis of NASH is influenced by factors such as insulin resistance, dyslipidemia and inflammatory pathways. Combination of drugs to counter these disease mechanisms will be an effective approach in the management of NASH.

NAFLD has become a growing public health problem with no licensed therapeutic agents. The cornerstone of current management is dietary and lifestyle intervention to achieve weight loss, along with the optimization of metabolic risk factors, such as diabetes mellitus and dyslipidemia. However, these goals are difficult to implement mainly because of poor adherence. Therefore, in selected cases, the off- label use of medications with demonstrated effects on NASH histological features can be considered.

Therefore, Inventors of the present invention found through research that the combination of sterol absorption inhibitor(s) with PPAR agonist(s) can significantly enhance the effect of PPAR agonist in improving non-alcoholic fatty liver disease (NAFLD) and/ or non-alcoholic steatohepatitis (NASH). It was found through experiments that PPAR agonist(s) and sterol absorption inhibitor(s) have a synergistic effect. In particular, the combination of pioglitazone and ezetimibe has shown unexpected results in the treatment of NAFLD and/or NASH. OBJECT OF THE PRESENT INVENTION

Another object of the present invention is to provide a pharmaceutical combination comprising an effective amount of peroxisome proliferator-activated receptor (PPAR) agonist(s) and an effective amount of sterol absorption inhibitor(s).

An object of the present invention is to provide a pharmaceutical combination for treatment or prevention of non-alcoholic fatty liver disease (NAFLD) and/or nonalcoholic steatohepatitis (NASH).

Another object of the present invention is to provide a pharmaceutical combination for treating or prevention of NAFLD and/or NASH wherein said combination comprising an effective amount of PPAR agonist(s) and an effective amount of sterol absorption inhibitor(s).

Another object of the present invention is to provide a method of treating or prevention of NAFLD and/or NASH by administering therapeutically effective amount of PPAR agonist(s) and therapeutically effective amount of sterol absorption inhibitor(s) either simultaneously/concurrently or altemately/sequentially.

Another object of the present invention is to provide a pharmaceutical composition for treating or prevention of NAFLD and/or NASH and other related disorders, wherein said composition comprising an effective amount of PPAR agonist(s), an effective amount of sterol absorption inhibitor(s) and optionally pharmaceutically acceptable excipient(s).

SUMMARY OF THE INVENTION

The present invention relates to pharmaceutical combination of peroxisome proliferator-activated receptor (PPAR) agonist(s) with sterol absorption inhibitor(s), as well as to the use of these combinations for treating and/or preventing non-alcoholic fatty liver disease (NAFLD) and/or non-alcoholic steatohepatitis (NASH) and other related disorders. Accordingly, main aspect of the present invention is to provide a pharmaceutical combination comprising: a) at least one PPAR agonist, or metabolites, isomers, polymorphs, pharmaceutically acceptable salts, esters, solvates thereof; and b) at least one sterol absorption inhibitor, or metabolites, isomers, polymorphs, pharmaceutically acceptable salts, esters solvates thereof.

According to the another aspect, the present invention provides the use of the aforementioned pharmaceutical combination in the preparation of medicines for the prevention and/or treatment of NAFLD and/or NASH and other related disorders including diabetes or cardiovascular diseases.

According to the another aspect of the present invention, the present invention provides a method of prevention and/or treatment of NAFLD and/or NASH by administering: a) therapeutically effective amount of at least one PPAR agonist, or metabolites, isomers, polymorphs, pharmaceutically acceptable salts, esters, solvates thereof; and b) therapeutically effective amount of at least one sterol absorption inhibitor, or metabolites, isomers, polymorphs, pharmaceutically acceptable salts, esters, solvates thereof.

According to the another aspect, the present invention provides a method of prevention and/or treatment of NAFLD and/or NASH by administering: a) therapeutically effective amount of at least one PPAR agonist, or metabolites, isomers, polymorphs, pharmaceutically acceptable salts, esters, solvates thereof; and b) therapeutically effective amount of at least one sterol absorption inhibitor, or metabolites, isomers, polymorphs, pharmaceutically acceptable salts, esters, solvates thereof; wherein said PPAR agonist is administered in an amount of about 5-80mg per day and said sterol absorption inhibitor is administered in an amount of about 2-20mg per day. According to another aspect, the present invention provides a method of treating or prevention of NAFLD and/or NASH by administering therapeutically effective amount of PPAR agonist(s) and therapeutically effective amount of sterol absorption inhibitor(s) either simultaneously/concurrently or altemately/sequentially.

According to another aspect, the present invention relates to pharmaceutical combinations comprising fixed dose of Pioglitazone and Ezetimibe for treating NAFLD and/or NASH and other related disorders, wherein Pioglitazone is in an amount of about 5-80mg, and Ezetimibe is in an amount of about 2-20mg.

According to another aspect, the present invention relates to pharmaceutical composition comprising Pioglitazone, Ezetimibe and one or more pharmaceutically acceptable excipients.

According to another aspect, the present invention relates to pharmaceutical composition comprising:

(a) at least one (one or more) PPAR agonist, such as but not limited to, peroxisome proliferator-activated receptor gamma including insulin sensitizers or thiazolidinedione compounds; or pharmaceutically acceptable salts or solvates thereof, or prodrugs of the at least one peroxisome proliferator-activated receptor agonist or of the salts or solvates thereof;

(b) one or more substituted azetidinone sterol absorption inhibitors, or pharmaceutically acceptable salts or solvates thereof, or prodrugs of the at least one sterol absorption inhibitor or of the salts or solvates thereof; and

(c) pharmaceutically acceptable excipients.

According to another aspect, the present invention relates to pharmaceutical composition comprising fixed dose of Pioglitazone and Ezetimibe for treating NAFLD and/or NASH and other related disorders, wherein Pioglitazone is in an amount of about 5-80mg, and Ezetimibe is in an amount of about 2-20mg. DETAILED DESCRIPTION OF THE INVENTION

Drawings:

Fig, 1A and IB: Effect of Pioglitazone and Ezetimibe combination on the Hepatic Triglycerides (A) and Plasma Triglyceride (B) of streptozotocin and high fat diet induced NASH mouse model

Fig, 2A and 2B: A dose dependent improvement in the NAFLD activity score in the combination groups compared to disease control observed from H&E staining model

The term "therapeutically effective amount" means that amount of a therapeutic agent of the composition, such as the peroxisome proliferator-activated receptor (PPAR) agonist(s), sterol absorption inhibitor(s) and other pharmacological or therapeutic agents described below, that will elicit a biological or medical response of a tissue, system, animal or mammal that is being sought by the administrator (such as a researcher, doctor or veterinarian) which includes alleviation of the symptoms of the condition or disease being treated and the prevention, slowing or halting of progression of one or more conditions, for example non-alcoholic fatty liver disease (NAFLD) and/or non-alcoholic steatohepatitis (NASH) and other related disorders such as vascular conditions, like hyperlipidaemia (for example atherosclerosis, hypercholesterolemia or sitosterolemia), vascular inflammation, stroke, diabetes, obesity and/or to reduce the level of sterol(s) (such as cholesterol) in the plasma.

As used herein, “pharmaceutical combination” means the administration of two or more therapeutic agents, such as PPAR agonist(s) and sterol absorption inhibitor(s), to prevent or treat a condition, for example NAFLD and/or NASH and other related disorders such as vascular condition, like hyperlipidaemia (for example atherosclerosis, hypercholesterolemia or sitosterolemia), vascular inflammation, stroke, diabetes, obesity and/or reduce the level of sterol(s) (such as cholesterol) in the plasma. As used herein, "vascular" comprises cardiovascular, cerebrovascular and combinations thereof. The pharmaceutical combinations and treatments of the present invention can be administered by any suitable means which produce contact of these compounds with the site of action in the body, for example in the plasma, liver or small intestine of a mammal or human. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single tablet or capsule having a fixed ratio of active ingredients or in multiple, separate capsules or dosage form for each therapeutic agent. Also, such administration includes use of each type of therapeutic agent in a sequential manner. In either case, the treatment using the pharmaceutical combination of present invention will provide beneficial effects in treating the condition. A potential advantage of the pharmaceutical combination disclosed herein may be a reduction in the required amount of an individual therapeutic compound or the overall total amount of therapeutic compounds that are effective in treating the condition. By using a combination of therapeutic agents, the side effects of the individual compounds can be reduced as compared to a monotherapy. The pharmaceutical combinations and treatments of the present invention can be administered wherein the co-administration of these therapeutic agents in a specific manner provides synergistic effects in treating NAFLD and/or NASH.

The pharmaceutical combination according to the embodiment of the present invention includes: at least one selected from the group comprising of PPAR agonist, or metabolites, isomers, polymorphs, pharmaceutically acceptable salts, esters, solvates thereof; and at least one selected from the group comprising of sterol absorption inhibitor(s), or metabolites, isomers, polymorphs, pharmaceutically acceptable salts, esters, solvates thereof.

According to the another embodiment of the present invention, the present invention provides the use of the aforementioned pharmaceutical combination in the preparation of medicines for the prevention and/or treatment of for NAFLD and/or NASH and other related disorders. In an another embodiment, the present invention provides a method of treating or prevention of NAFLD and/or NASH by administering therapeutically effective amount of PPAR agonist(s) and therapeutically effective amount of sterol absorption inhibitor(s) either simultaneously/concurrently or altemately/sequentially.

In an another embodiment, the present invention provides a pharmaceutical combination comprising: a) at least one PPAR agonist, or metabolites, isomers, polymorphs, pharmaceutically acceptable salts, esters, solvates thereof, wherein said PPAR agonist is present in an amount of about 5-80mg; and b) at least one sterol absorption inhibitor(s), or metabolites, isomers, polymorphs, pharmaceutically acceptable salts, esters, solvates thereof, wherein said sterol absorption inhibitor is present in an amount of about 2-20mg.

In an another embodiment, the present invention provides a method of prevention and/or treatment of NAFLD and/or NASH by administering: a) therapeutically effective amount of at least one PPAR agonist or metabolites, isomers, polymorphs, pharmaceutically acceptable salts, esters, solvates thereof; and b) therapeutically effective amount of at least one sterol absorption inhibitor(s) or, metabolites, stereoisomers, polymorphs, pharmaceutically acceptable salts, solvates thereof; wherein said PPAR agonist is administered in an amount of about 5-80mg per day and said sterol absorption inhibitors is administered in an amount of about 2-20mg per day.

In an another embodiment, the present invention provides a method of prevention and/or treatment of NASH in patients having diabetes, cardiovascular disease and other related disorders, by administering therapeutically effective amount of at least one PPAR agonist and therapeutically effective amount of atleast one sterol absorption inhibitor optionally with pharmaceutically acceptable excipient. The pharmaceutical combination as per the present invention can be given in a single dose or as divided doses administered at appropriate intervals, e.g. as two, three, four or more sub-doses per patient per day, with or without food.

In one another embodiment, the present invention further provides a pharmaceutical composition comprising at least one PPAR agonist and at least one sterol absorption inhibitor and optionally one or more pharmaceutically acceptable excipients, for treating NAFLD and/ or NASH and other related disorders. Both the drugs can be administered in a single dosage form or each in separate dosage forms, or they can be administered by different routes.

The pharmaceutical combinations and/or compositions of the present invention comprise at least one (one or more) agonist of peroxisome proliferator-activated receptors (PPAR). These receptors act as agonists for the peroxisome proliferator- activated receptors. Three subtypes of PPAR have been identified, and these are designated as peroxisome proliferator-activated receptor alpha (PPARa), peroxisome proliferator-activated receptor gamma (PPARy) and peroxisome proliferator-activated receptor delta (PPAR5). It should be noted that PPAR5 is also referred to in the literature as NUCI, and each of these names refers to the same receptor.

PPARa regulates the metabolism of lipids. PPARa is activated by fibrates and a number of medium and long-chain fatty acids, and it is involved in stimulating p- oxidation of fatty acids. The PPARy receptor subtypes are involved in activating the program of adipocyte differentiation and are not involved in stimulating peroxisome proliferation in the liver. PPAR has been identified as being useful in increasing high density lipoprotein (HDL) level in humans.

In another embodiment, the PPAR agonist are identified as three subtypes such as PPARa, PPARy and PPAR5, wherein present invention specifically provides a pharmaceutical combination and/or composition comprising PPARy activators or agonists selected from glitazones or thiazolidinedione such as troglitazone, pioglitazone, rosiglitazone, ciglitazone, englitazone, darglitazone, and preferably pioglitazone.

In another embodiment, the sterol absorption inhibitor used in the present combination and/or composition is selected from ezetimibe, simvastatin, pravastatin, lovastatin, and preferably ezetimibe.

In specific embodiment, the present invention provides a pharmaceutical composition comprising therapeutically effective amount of Pioglitazone and therapeutically effective amount of Ezetimibe along with one or more pharmaceutically acceptable excipients, for treating NAFLD and/ or NASH and other related disorders.

In another specific embodiment, the present invention provides a pharmaceutical composition comprising therapeutically effective amount of Pioglitazone and therapeutically effective amount of Ezetimibe with one or more pharmaceutically acceptable excipients, wherein said Pioglitazone is in an amount of about 5-80mg, and said Ezetimibe is in an amount of about 2-2 Omg.

Briefly, and as described in more detail below, described herein are combinations, methods of making the said compositions and methods for treating NAFLD and/or NASH by administering Pioglitazone with Ezetimibe, specifically a fixed dose combination of Pioglitazone and Ezetimibe.

The peroxisome proliferator-activated receptor agonist(s) are administered in a therapeutically effective amount to treat the specified condition, for example in a daily dose can range from about 0.1 to about 1000 mg per day, preferably about 0.25 to about 100 mg/day, and more preferably about 5 to 80 mg per day, given in a single dose or divided doses. The exact dose, however, is determined by the attending clinician and is dependent on such factors as the potency of the compound administered, the age, weight, condition and response of the patient. Pioglitazone is an antidiabetic agent that selectively stimulates the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-y) and to a lesser extent PPAR-a. It modulates the transcription of the genes involved in the control of glucose and lipid metabolism in the muscle, adipose tissue, and the liver. As a result, pioglitazone reduces insulin resistance in the liver and peripheral tissues, decreases gluconeogenesis in the liver, and reduces quantity of glucose and glycated hemoglobin in the bloodstream. Overall, pioglitazone targets insulin resistance and adipose tissue dysfunction that causes liver lipotoxicity and inflammation in fatty liver disease. Pioglitazone improved steatosis, lobular inflammation, hepatocellular ballooning, fibrosis and NASH resolution, concluding it as the only agent that has shown consistent benefit and efficacy in a number of clinical trials.

At present, the main drugs for the treatment of NAFLD and/or NASH include insulin sensitizers (biguanides such as metformin, thiazolidinedione compounds such as rosiglitazone and pioglitazone), lipid-lowering drugs (statins, fibrates), choleretics Drugs (ursodeoxycholic acid), liver-protecting drugs (vitamin E, silymarin, acetylcysteine) and traditional Chinese medicines (polygonum multiflorum, salvia miltiorrhiza, Alisma, Ligusticum chuanxiong, cassia, hawthorn, etc.). At present, the main drugs for the treatment of NAFLD include insulin sensitizers (biguanides such as metformin, thiazolidinedione compounds such as rosiglitazone and pioglitazone), lipid-lowering drugs (statins, fibrates), choleretics Drugs (ursodeoxycholic acid), liver-protecting drugs (vitamin E, silymarin, acetylcysteine) and traditional Chinese medicines (polygonum multiflorum, salvia miltiorrhiza, Alisma, Ligusticum chuanxiong, cassia, hawthorn, etc.).

The pharmaceutical combinations of the present invention comprises one or more substituted azetidinone as sterol absorption inhibitors. As used herein, "sterol absorption inhibitor" means a compound capable of inhibiting the absorption of one or more sterols, including but not limited to cholesterol, phytosterols (such as sitosterol, campesterol, stigmasterol and avenosterol), 5a-stanols (such as cholestanol, 5a-campestanol, 5a-sitostanol), and mixtures thereof, when administered in a therapeutically effective (sterol absorption inhibiting) amount to a mammal or human.

The sterol (cholesterol) absorption inhibitor includes ezetimibe which has been shown to ameliorate NAFLD pathology in a combined clinical study with PPAR agonist such as Pioglitazone and in experimental animal models.

Ezetimibe combined with Pioglitazone inhibits the absorption of cholesterol from the small intestine and decreases the amount of cholesterol normally available to liver cells. The lower levels of cholesterol in the liver cells leads them to absorb more cholesterol from circulation and thus lowering the levels of circulating cholesterol. It blocks the critical mediator of cholesterol absorption, the Niemann- Pick Cl-like 1 (NPC1L1) protein on the gastrointestinal tract epithelial cells, as well as in hepatocytes; it blocks aminopeptidase and interrupts caveolin 1-annexin A2 complex involved in trafficking cholesterol.

In clinical study using ezetimibe and Pioglitazone, it was demonstrated that the said combination significantly reduce serum cholesterol and cholesterol absorption markers and also improved hepatic fibrosis, ballooning score in NAFLD patients. Ezetimibe is estimated to inhibit not only dietary and biliary cholesterol absorption through the small intestine, but also reabsorption of biliary cholesterol in the liver. Thus, ezetimibe may inhibit liver fibrosis by ameliorating cholesterol-induced activation of hepatic stellate cells in patients with NAFLD.

The sterol absorption inhibitor(s) are administered in a therapeutically effective amount to treat the specified condition, for example in a daily dose can range from about 0.1 to about 1000 mg per day, preferably about 0.25 to about 100 mg/day, and more preferably about 2 to 20 mg per day, given in a single dose or divided doses. The exact dose, however, is determined by the attending clinician and is dependent on such factors as the potency of the compound administered, the age, weight, condition and response of the patient. According to specific embodiments of the present invention, the above-mentioned pharmaceutical combinations/compositions, in addition to the aforementioned active ingredients, also include a pharmaceutically acceptable excipient(s). According to an embodiment of the present invention, the pharmaceutically acceptable excipient is selected from, but not limited to, pharmaceutically acceptable solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers/diluents, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrating agents, penetration enhancers, pH Value regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, opacifier, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retarder, a polymer skeleton material, and a film-forming material. Herein, the pharmaceutical composition is formulated into a form suitable for administration of clinical pharmaceutical preparations.

Suitable fillers/ diluents include, without limitation, starch, com starch, potato starch, pregelatinized starch, dry starch, disaccharides, lactose, cellulose, cellulose derivatives, such as silicified microcrystalline cellulose, microcrystalline cellulose, mannitol, sorbitol, xylitol, trehalose, colloidal silica, sucrose or other sugars or sugar derivatives, calcium hydrogen phosphate, dicalcium phosphate, low- substituted hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, and/or combinations thereof. When present, a filler may be employed in an amount ranging from about 10% to about 80%, preferably from about 20% to about 80% by weight of the formulation.

Suitable binders include, without limitation, microcrystalline cellulose, polyvinylpyrrolidone (PVP), such as e.g., PVP K 30 or PVP90F, polyethylene glycols (PEG), e.g., PEG 4000, hydroxypropylmethyl cellulose, hydroxypropyl cellulose, both preferably of medium to high viscosity, e.g. viscosity grades 3 or 6 cps, pregelatinized starch, copovidone, gelatin, sugars and/or combinations thereof. When present, a binder may be employed in an amount ranging from about 0.1% to about 20%, preferably from about 0.5% to about 15%, such as 1% to 10%, by weight of the formulation.

Suitable lubricants include, without limitation, zinc stearate, magnesium stearate, sodium stearyl fumarate, aluminium or calcium silicate, stearic acid, PEG, talc and/or combinations thereof. When present, a lubricant may be employed in an amount ranging from about 0.01% to about 10%, preferably from about 0.1% to about 5%, by weight of the formulation.

Suitable disintegrants include, without limitation, carboxymethylcellulose calcium (CMC-Ca), carboxymethylcellulose sodium (CMC -Na), crosslinked PVP (e.g. crospovidone, polyplasdone XL or kollidon CL), croscarmellose sodium, alginic acid, sodium alginate and guar gum, most preferably crosslinked PVP (crospovidone), crosslinked CMC (Ac-Di-Sol), carboxymethyl starch-Na (pirimojel and explotab), sodium starch glycolate, low-substituted hydroxypropyl cellulose, polacrillin and/or combinations thereof. A disintegrant is employed in an amount of 0.01 to 15%, such as of 0.05 to 12%, such as at least 0.1 to 10%, by weight of the formulation.

Suitable glidants include, without limitation, zinc stearate, colloidal silicon dioxide (e.g., Aerosil 200), magnesium trisilicate, powdered cellulose, starch, talc and/or combinations thereof. When present, a glidant may be employed in an amount ranging from about 0.01% to about 10%, preferably from about 0. 1% to about 5%, by weight of the formulation.

The surfactants include but are not limited to anionic, cationic, non-ionic or amphoteric surfactants or those known to the person skilled in the art. Suitable surface active are poloxamer, polysorbate, cremophore, soluplus, lecithin and sodium lauryl sulfate and/or combinations thereof. The pharmaceutical combination/composition of the present invention may be further film coated. Film coating may be an immediate release or an extended release coating. The extended release coating comprises at least one or more extended release polymer and one or more pharmaceutically acceptable excipients.

Suitable film forming agents include, for example, polyvinylpyrrolidone, natural gums, starches, and cellulosic polymers. Examples of cellulosic polymers include, but are not limited to, polyvinyl alcohol, hydroxypropyl methyl cellulose ("HPMC"), carboxymethyl cellulose ("CMC") or salts thereof, hydroxypropyl cellulose ("HPC"), methylcellulose ("MC"), hydroxyethyl cellulose ("HEC"), ethylcellulose, acrylates, Eudragits, and the like. Further, commercially available coating materials are available marketed under the brand name Opadry®. The polymers such as polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl acetate can be used as a film forming agent. In one aspect, the film forming agent can be used in an amount from l%to 10% by weight of the composition.

Coloring agents include any FDA approved color for oral use.

Suitable plasticizers are selected from the group comprising of triethylcitrate, dibutyl sebacate, acetylated triacetin, tributylcitrate, glycerlotributyrate, monoglyceride, olive oil, sesame oil, acetyltributylcitrate, acetyltriethylcitrate, glycerin, sorbitol, diethyl oxalate, diethyl phthalate, diethyl malate, diethyl fumarate, dibutyl succinate and/or combinations thereof.

Suitable opacifiers are selected from the group comprising of titanium dioxide, manganese dioxide, iron oxide, silicon dioxide and/or combinations thereof.

According to a specific embodiment of the present invention, the above-mentioned pharmaceutical combination/composition may be any dosage form suitable for oral administration. When administered orally, the pharmaceutical dosage form of the present invention using the pharmaceutical combination/composition as the active ingredient includes, but is not limited to, tablets, sublingual tablets, effervescent tablets, coated tablets, sugar-coated tablets, dispersible tablets, enteric -coated tablets, granules, gelatin capsules, soft gelatin capsules, enteric-coated capsules, sustained-release capsules, controlled-release capsules, oral liquids, preferably tablets or capsules.

Accordingly, in another embodiment, the present invention provides pharmaceutical composition comprising:

(a) at least one PPAR agonist selected from troglitazone, pioglitazone, rosiglitazone, ciglitazone, englitazone, darglitazone;

(b) at least one sterol absorption inhibitor selected from ezetimibe, simvastatin, pravastatin, lovastatin; and

(c) one or more pharmaceutically acceptable excipients selected from binder, disintegrant, lubricant and diluent.

In an embodiment, the present invention is directed to pharmaceutical combinations or compositions, kits and methods of treatment using the same comprising;

(c) pharmaceutically acceptable excipients.

In another embodiment, the pharmaceutical combination of the present invention can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. According to specific embodiment, the present invention relates to combinations comprising fixed dose of Pioglitazone and Ezetimibe for treatment of NAFLD and/or NASH and other related disorders.

According to another specific embodiment, the present invention relates to pharmaceutical combination of Pioglitazone and Ezetimibe wherein said combination further comprises of pharmaceutically acceptable excipients.

According to another specific embodiment, the present invention provides a pharmaceutical combination comprising Pioglitazone and Ezetimibe, wherein Pioglitazone and Ezetimibe are present in a single dosage form or in separate dosage forms.

According to another specific embodiment, the present invention provides a pharmaceutical composition comprising Pioglitazone and Ezetimibe, wherein Pioglitazone and Ezetimibe are present in a single dosage form or in separate dosage forms.

In other embodiments, the present invention relates to a kit for conveniently and effectively carrying out the methods in accordance with the present invention. In general, the pharmaceutical pack or kit comprises one or more containers filled with one or more of the ingredients of the pharmaceutical compositions/ combinations of the invention. Such kits are especially suited for the delivery of solid oral forms such as tablets or capsules. Such a kit preferably includes a number of unit dosages, and may also include a card having the dosages oriented in the order of their intended use. If desired, a memory aid can be provided, for example in the form of numbers, letters, or other markings or with a calendar insert, designating the days in the treatment schedule in which the dosages can be administered. Alternatively, placebo dosages, or calcium dietary supplements, either in a form similar to or distinct from the dosages of the pharmaceutical compositions, can be included to provide a kit in which a dosage is taken every day. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

In certain embodiment, the pharmaceutical combination of the present invention which is present as a separate or multiple dosage form, preferably as a kit, is useful in combination therapy to flexibly suit the individual therapeutic needs of the patient.

Accordingly in an embodiment, the present invention provides a kit comprising pharmaceutical combination comprising two components wherein said components includes: a) first component comprising at least one PPAR agonist, or metabolites, isomers, polymorphs, pharmaceutically acceptable salts, esters, solvates thereof; and b) second component comprising at least one sterol absorption inhibitor(s), or metabolites, isomers, polymorphs, pharmaceutically acceptable salts, esters, solvates thereof.

EXAMPLES

Evaluating the effect of Pioglitazone and Ezetimibe Combination in Non- Alcoholic Steatohepatitis (NASH) Mouse model.

EXPERIMENTAL DESIGN:

Male Neonatal C57BL/6 mice of age 2 days weeks were used for the study. During this period, the mice were observed daily for clinical signs. On day 1 of study each neonatal mouse was administered with Streptozotocin 200pg/mouse subcutaneously. On 4th week onwards of the study rodent diet with 60 kcal% Fat (Cat # D 12492, Research Diets Inc.) was provided to all the study animals except G1 Naive control (provided with standard -Normal pellet diet). On 6th week animals were randomized on basis of body weight and blood glucose level to different treatment group. Gl, untreated, G2, disease control administered with vehicle, G3 (Pioglitazone, 15mg/kg, po, QD and Ezetimibe, 10mg/kg,po, QD), G4 (Pioglitazone, 15mg/kg, p.o., QD and Ezetimibe, 5mg/kg, p.o., QD), G5 (Pioglitazone, lOmg/kg, p.o., QD and Ezetimibe, lOmg/kg, p.o., QD), G6 (Pioglitazone, lOmg/kg, p.o., QD and Ezetimibe, 5mg/kg, p.o., QD) and G7 was treated with Pioglitazone hydrochloride (30 mg/kg, p.o., QD).

Treatment of vehicle, test compounds and Pioglitazone hydrochloride was continued for 6 weeks i.e. up to 12 week of study. During the study the body weight of animals was recorded twice a week.

Example 1: Plasma and Hepatic Triglyceride Levels

Experiment: At the end of study (12th week), blood was collected by retro-orbital plexus under isoflurane anaesthesia. Plasma was separated and collected for evaluating plasma triglyceride levels. All the animals were humanely sacrificed by carbon dioxide asphyxiation. Also liver was collected and weighed. Liver sample was further processed for the hepatic cholesterol and triglyceride levels estimation. Plasma and Hepatic cholesterol was estimated by using appropriate assay kit. All the estimation was conducted on Fully Auto Biochemistry analyzer. Data were presented as the mean ± SEM (n=8). Statistical analysis was done with GraphPad Prism-5 using one way ANOVA followed by Dunnett's test/Tukey’s post hoc test and two way ANOVA followed by Bonferroni post hoc test. Data was considered statistically significant, if P value is less than 0.05.

Results:

Effect of combination of pioglitazone and ezetimibe was analysed in NASH mouse model at different doses. There was no effect on the food and water intake in all groups. Pioglitazone, 15 and lOmg/kg was assessed for efficacy in combination with ezetimibe at lOmg/kg and 5 mg/kg doses. Pioglitazone, 30mg/kg was considered as comparator molecule. A dose dependent reduction of plasma and hepatic triglycerides levels were observed in the animals treated with pioglitazone and ezetimibe. Combination of pioglitazone (lOmg/kg) and ezetimibe (5 or lOmg/kg) significantly reduced plasma and hepatic triglycerides compared to disease control and the reduction was comparable to pioglitazone, 30 mg/kg (Figure 1A and IB). Also, it was observed that combination of pioglitazone (15mg/kg) and ezetimibe (5 or lOmg/kg) significantly reduced plasma and hepatic triglycerides as compared to disease control and the reduction was comparable to pioglitazone, 30 mg/kg.

Example 2: Histological Analysis

Experiment: At end of study on 12th week, liver was collected and weighed. Further the liver was processed for histopathology score (H & E Staining) and Ascrosft score (MT Staining). All the animals were humanely sacrificed by carbon dioxide asphyxiation.

Histology was performed for liver samples using MT (Masson's Trichrome) staining for Ashcroft score and Hematoxylin and Eosin (H&E) staining for inflammatory and fibrosis markers. Briefly, liver from all the experimental groups were fixed in 10% buffered neutral formalin for one week at room temperature. Then tissues were dehydrated in a graded series of alcohol, cleaned in xylene and then embedded in paraffin. Serial sections of 4-mm-thickness were prepared from each tissue embedded paraffin blocks using a rotary microtome and employed for histological examination using light microscope after being stained by a MT & hematoxylin and eosin procedure. Blind histopathological evaluation of samples was performed by the pathologist. All data will be expressed as the mean ± SEM. Data on each parameter will be summarized in tabular form. Appropriate graphical representation will be done using suitable method. Statistical analysis will be done with GraphPad Prism-5 using one way ANOVA followed by Dunnett's test/Tukey’s post hoc test and/or two way ANOVA followed by Bonferroni post hoc test. Data will be considered statistically significant, if P value is less than 0.05.

Results:

Histological analysis of livers by hematoxylin and eosin (H & E) staining as well as masson’s trichrome staining showed a significant disease induction in disease control compared to naive control livers. A dose dependent improvement (G5 and G6) in the NAFLD activity score in the combination groups compared to disease control was observed from H&E staining (Figure 2A). Similarly, histopathology analysis performed by massons’s trichrome staining of liver sections, which is a method to identify the collagen accumulation, also showed a dose dependent improvement (G5 and G6) in the combination group of animals compared to disease control (Figure 2B). Overall, a dose dependent improvement in the NAS score was observed with combinations of pioglitazone and ezetimibe. The combinations showed a significant improvement in the NAS score compared to disease control and also the improvement was comparable to group treated with pioglitazone alone.

Our findings suggest that combination therapy of pioglitazone and ezetimibe improved plasma liver markers and plasma triglycerides. Combination therapy showed equal or numerically superior efficacy in terms of NAS as well as fibrosis score compared to pioglitazone in streptozotocin and high fat diet induced NASH mouse model

Example 3: Composition of Ezetimibe and Pioglitazone

Preparation:

Ezetimibe, Pioglitazone, lactose monohydrate, hydroxypropyl cellulose, colloidal silicon dioxide, and magnesium stearate are mixed together and punched or filled into tablets followed by film coating.

Example 4: Monolayer Tablet of Ezetimibe and Pioglitazone

Preparation:

Ezetimibe, Pioglitazone, lactose monohydrate, hydroxypropyl cellulose, colloidal silicon dioxide, and magnesium stearate are mixed together and punched into tablets followed by film coating.

Example 5: Bilayer Tablet of Ezetimibe and Pioglitazone

Preparation: Ezetimibe, lactose monohydrate, sodium starch glycolate, povidone, colloidal silicon dioxide and magnesium stearate are mixed together and punched into an Ezetimibe layer over which is punched Pioglitazone layer which is prepared by mixing Pioglitazone, anhydrous lactose, microcrystalline cellulose, crospovidone, silicon dioxide and magnesium stearate and then punching into a Pioglitazone layer. The bilayer tablet is then film coated.

Claims

24 CLAIMS

1. A pharmaceutical combination comprising: a) at least one PPAR agonist, or metabolites, isomers, polymorphs, pharmaceutically acceptable salts, esters, solvates thereof; and b) at least one sterol absorption inhibitor, or metabolites, isomers, polymorphs, pharmaceutically acceptable salts, esters solvates thereof.

2. The pharmaceutical combination as claimed in claim 1, wherein said combination is administered either simultaneously/concurrently or altemately/sequentially.

3. The pharmaceutical combination as claimed in claim 1, wherein said combination is used for the treatment of NAFLD and/or NASH and other related disorders.

4. The pharmaceutical combination as claimed in claim 1, wherein said PPAR agonist is selected from troglitazone, pioglitazone, rosiglitazone, ciglitazone, englitazone, and darglitazone; and wherein said sterol absorption inhibitor is selected from ezetimibe, simvastatin, pravastatin, and lovastatin.

5. The pharmaceutical combination as claimed in claim 4, wherein said PPAR agonist is Pioglitazone, and wherein said sterol absorption inhibitor is Ezetimibe.

6. The pharmaceutical combination as claimed in claim 1, wherein said PPAR agonist is in an amount ranging from 5 to 80 mg, given in a single dose or divided doses, and wherein said sterol absorption inhibitor is in an amount ranging from 2 to 20 mg, given in a single dose or divided doses.

7. A pharmaceutical combination comprising: a) at least one PPAR agonist selected from troglitazone, pioglitazone, rosiglitazone, ciglitazone, englitazone, and darglitazone, wherein said PPAR agonist is present in an amount of about 5-80mg; and b) at least one sterol absorption inhibitor selected from ezetimibe, simvastatin, pravastatin, and lovastatin, wherein said sterol absorption inhibitor is present in an amount of about 2-20mg.

8. A pharmaceutical composition comprising:

(a) at least one PPAR agonist selected from peroxisome proliferator-activated receptor gamma including insulin sensitizers or thiazolidinedione compounds; or pharmaceutically acceptable salts or solvates thereof, or prodrugs of the at least one peroxisome proliferator-activated receptor agonist or of the salts or solvates thereof;

(c) pharmaceutically acceptable excipients.

9. The pharmaceutical composition as claimed in claim 8, wherein said composition comprising:

10. The pharmaceutical composition as claimed any of the preceding claims, wherein said PPAR agonist is in amount of about of 5 to 80 mg, and wherein said sterol absorption inhibitor is in amount of about 2 to 20 mg.

11. The pharmaceutical composition as claimed in claim 9, wherein said PPAR agonist is pioglitazone, and wherein said sterol absorption inhibitor is ezetimibe.

12. The pharmaceutical composition as claimed in claim 8, wherein said pharmaceutically acceptable excipient is selected from pharmaceutically acceptable solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers/diluents, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrating agents, penetration enhancers, pH Value regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, opacifier, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retarder, a polymer skeleton material, a film-forming material, and mixture thereof.

13. A kit comprising pharmaceutical combination comprising two components wherein said components includes: a) first component comprising at least one PPAR agonist, or metabolites, isomers, polymorphs, pharmaceutically acceptable salts, esters, solvates thereof; and b) second component comprising at least one sterol absorption inhibitor(s), or metabolites, isomers, polymorphs, pharmaceutically acceptable salts, esters, solvates thereof.

14. The kit as claimed in claim 13, wherein said sterol absorption inhibitor is in a therapeutically effective amount ranging from 2 to 20 mg, given in a single dose or divided doses.

15. The kit as claimed in claim 13, wherein said PPAR agonist is in a therapeutically effective amount ranging from 5 to 80 mg, given in a single dose or divided doses.

16. A method of treatment of NAFLD and/or NASH by administering: a) therapeutically effective amount of at least one PPAR agonist, or metabolites, isomers, polymorphs, pharmaceutically acceptable salts, esters, solvates thereof; and b) therapeutically effective amount of at least one sterol absorption inhibitor, or metabolites, isomers, polymorphs, pharmaceutically acceptable salts, esters, solvates thereof. 27

17. The method of treatment as claimed in claim 16, wherein said method of treatment comprises administering: a) therapeutically effective amount of at least one PPAR agonist, or metabolites, isomers, polymorphs, pharmaceutically acceptable salts, esters, solvates thereof; and b) therapeutically effective amount of at least one sterol absorption inhibitor, or metabolites, isomers, polymorphs, pharmaceutically acceptable salts, esters , solvates thereof; wherein said PPAR agonist is administered in an amount of about 5-80mg per day and said sterol absorption inhibitor is administered in an amount of about 2-20mg per day.

18. The method of treatment as claimed in 17, wherein said PPAR agonist is selected from troglitazone, pioglitazone, rosiglitazone, ciglitazone, englitazone, darglitazone, and wherein said sterol absorption inhibitor is selected from ezetimibe, simvastatin, pravastatin, and lovastatin.

19. The method of treatment as claimed in 18, wherein said PPAR agonist is pioglitazone, and wherein said sterol absorption inhibitor is ezetimibe.

20. Use of pharmaceutical composition comprising atleast one PPAR agonist, atleast one sterol absorption inhibitor along with atleast one pharmaceutically acceptable excipient, for the treatment of NAFLD and/or NASH, wherein said PPAR agonist is in an amount of about 5-80mg and said sterol absorption inhibitor is in an amount of about 2-2 Omg.