FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13]
1. Title of the invention: "NOVEL COMBINATION FOR THE TREATMENT OF NONALCHOLIC FATTY LIVER DISEASE"
2. Macleods Pharmaceuticals Ltd., an Indian Company, having its Registered Office at 304 - Atlanta Arcade, Opp. Leela Hotel, Marol Church Road, Andheri (East), Mumbai - 400 059, Maharashtra, India.
3. The following specification particularly describes the invention and the manner in which it is to be performed.

FIELD OF THE INVENTION
The present invention relates to a pharmaceutical compositions comprising novel combination of Vitamin E, Fraxinus excelsior and/or N-acetyl-L-cysteine (NAC) as active ingredients in a single unit dosage form for the prevention and/or treatment of non-alcoholic fatty liver disease (NAFLD).
BACKGROUND OF THE INVENTION
Since first described by Ludwig in 1980, non-alcoholic fatty liver disease (NAFLD) has progressed from a poorly understood liver disease to one with more well defined boundaries. Nonalcoholic fatty liver disease is defined as a disease with fat deposition in the liver, which occurs in patients whose alcohol ingestion history is not long enough to cause liver injury, except for cases of known etiology, such as viral hepatitis and autoimmune hepatitis. NAFLD has close association with obesity, insulin resistance and dyslipidemia, therefore it is considered as the hepatic manifestation of the metabolic syndrome. NAFLD includes a wide spectrum of clinico-pathologic entities ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), with the possibility of progression to cirrhosis and hepatocellular carcinoma was described in Ludwig J et al., Mayo Clin Proc. 1980, 55(7), 434-438. Steatosis is an accumulation of fat in the liver. When this progresses to become associated with inflammation, it is known as steatohepatitis. In conclusion, the pathogenesis of NAFLD appears to involve a multi-hit process. The first hit is the steatosis which is believed to be triggered by insulin resistance, and the second hit, which involves cytokines alteration and oxidative stress, results in disease progression.
In view of the high prevalence of disorders associated with hepatic lipid deposits, the severity of these conditions, and the lack of proven treatments, it is important to develop new treatments for such conditions.

SUMMARY OF THE INVENTION
The present invention relates to a pharmaceutical composition comprising combination of Vitamin E, Fraxinus excelsior and/or N-acetyl-L-cysteine (NAC) as active ingredients in a single unit dosage form for the prevention and/or treatment of fatty liver disease particularly non-alcoholic fatty liver disease (NAFLD).
Further advantages and embodiments of the present invention will become apparent from the following description.
DETAILED DESCRIPTION OF THE INVENTION
The prevalence of non-alcoholic fatty liver disease is likely to increase over the time due to the epidemics of obesity and diabetes. Presently, there is no definitive treatment for NAFLD.
Oxidative stress plays a central role in the transition from simple steatosis to nonalcoholic steatohepatitis. An effective therapeutic strategy is to target reduction in oxidative stress in NAFLD patients. Oxidative stress is considered a major contributor as the "second hit" in the pathogenesis of NAFLD and NASH, justifying the study of several antioxidants in NAFLD treatment.
It is therefore apparent that investigating compounds able to counteract this oxidative stress may have a relevant clinical impact. Balanced human diets contain multiple antioxidants and there is strong evidence that additive and synergistic interactions occur among those antioxidant substances. Under a clinical perspective, this suggests that the use of combination compositions containing multiple substances with antioxidant properties has the potential to provide a significantly better protection against oxidative stress than the use of each single antioxidant alone.

Antioxidants such as Vitamin E and N-acetyl-cysteine (NAC) have also been studied for their purported antifibrotic effects. Alpha-tocopherol, the form of Vitamin E that is preferentially metabolized in humans, inhibits transforming growth factor beta-1, which is thought to contribute to fibrosis progression.
Vitamin E is a powerful antioxidant, which can improve the impact of free radicals on the body in the normal process of metabolism and oxidative stress. Studies have shown that Vitamin E can interrupt the radical chain reaction, prevent free radicals into the cell membrane, and maintain the stability of cell membranes. Vitamin E inhibits the generation of mitochondrial reactive oxygen species (ROS), through increasing the mitochondrial membrane potential and improving mitochondrial function. Meanwhile, it can protect polyunsaturated fatty acids in the cell membrane, and inhibit lipid peroxidation. Clinical trials also showed that Vitamin E can improve the extent of oxidative stress in patients with NASH. Vitamin E is an effective antioxidant, which could relieve NAFLD symptoms by improving the balance of oxidation and anti-oxidation. However, recent researches indicate that the functional mechanisms of Vitamin E are not only limited to anti-oxidation, but also include adjusting the metabolism disorders of glucose and lipid. Hence, Vitamin E is an obvious choice once the role of oxidative stress was made clear in NAFLD. The combination therapy with Pioglitazone and Vitamin E is superior to Vitamin E alone for the treatment of NASH is disclosed in Sanyal et al., 2004, Clinical gastroenterology and hepatology, 2, 1107-1115. Thus, combination therapy is proposed to be more effective than monotherapy in the treatment of NAFLD.
N-acetyl-L-cysteine (NAC) is an amino-acid derivative (small protein), water-soluble, thiol-containing antioxidant that has been used both experimentally and clinically since the 1950s. It was originally used as a mucolytic agent in congestive and obstructive lung diseases such as cystic fibrosis and chronic bronchitis, diseases characterized by hypersecretion of respiratory mucus. More recently, it has been used for treatment of acetaminophen overdose.

Experimentally, it has been used as an antioxidant, in particular to decrease damage to cell components by oxidants. NAC restores intracellular levels of glutathione, which is the body's most powerful antioxidant that helps the liver to protect against toxicity, and it is most needed by those with liver disease.
Fraxinus excelsior, Linne, known as bird's tongue, weeping ash, European ash, common ash or simply ash is a species of Fraxinus that grows in temperate regions and is native to most of Europe & Asia. The Fraxinus species have been used as food and condiments as well as traditional medicine due to several claimed beneficial biological activities including antioxidative, anti¬inflammatory, analgesic, antipyretic, antirheumatic, antiobesity, antidiabetic as well as hypotensive properties. European Medicine Agency (EMA) final assessment report on Fraxinus excelsior L. or Fraxinus angustifolia Vahl, folium discloses powdered herbal substance of dried leaf of Fraxinus excelsior L. or Fraxinus angustifolia Vahl or hybrids of these two species or of a mixture in capsules. Also discloses combination of herbal substance(s) and/or herbal preparation(s) including a description of vitamin(s) and/or mineral(s) as ingredients of traditional combination herbal medicinal products assessed, where applicable. The studies on a patented seed extract of F. excelsior, marketed as FraxiPure™, have confirmed the ability of this seed extract to reduce glycemia both in vivo and in acute clinical study is disclosed in Visen et al., 2009, J. Ethnopharmacol. 126, 226-232. Mice fed a high fat diet supplemented with FraxiPure™ displayed significant reductions in fasting glucose and fasting plasma insulin levels at study end compared to the high-fat diet control. Body weights were significantly decreased in mice fed FraxiPure™ compared to mice fed the high-fat diet for week 9 of the 20-week study. Administration of FraxiPure™ in the diet ameliorated incidences of fatty liver and reduced liver weight and plasma ALT induced by the high fat diet is disclosed in Ibarra et al, 2011, Phytomedicine 18, 479-485. The results suggested that extract itself was without adverse effects on the liver and that FraxiPure™ may possess hepatoprotective properties.

U.S. Patent No. 8293292 discloses a Fraxinus excelsior seed extract that can be administered for therapeutic treatment of a subject, including a human, by blocking fat synthesis, activating PPAR-alpha, increasing hypoglycemic activity, reducing bodyweight, controlling fasting plasma insulin levels against hyperinsulinemia, and promoting insulin sensitivity and causing a beneficial acute insulinotropic effect. The Fraxinus excelsior seed extract includes, inter alia, an isolated compound (2S,3E,4S)2H-Pyran-4-acetic acid-3-ethylidene-2-[(6-0-p-D-glucopyranosyl-P-D-glucopyranosyl)oxy]-3,4-dihydro-5-(methoxycarbonyl)methyl ester, commonly called excelside A, an isolated compound (2S,3E,4S) 2H-Pyran-4-acetic acid-3-ethylidene-2-[(6-0-P-D-glucopyranosyl-P-D-glucopyranosyl)oxy]-3,4-dihydro-5-(methoxycarbonyl)2-(4-hydroxyphenyl)ethyl ester, commonly called excelside B, and the compounds GI5, GI3, nuzhenide, and oleoside dimethyl ester.
Glucevia® is a marketed natural extract of Fraxinus excelsior L. derived from seeds/fruits standardized to 10% Nuzhenide and GI3 as its active ingredients as reported in U.S. Patent No. 8293292. Glucevia® (product code EA149251) was supplied by Naturex S.A. (Avignon, France). The nutritional profile and safety of the extract have been evaluated in a double-blind, placebo-controlled, randomized clinical study (Flanagan et al. 2013), demonstrating the safety and tolerability for consumption in healthy subjects, as observed in biochemical and hematological assessments. In addition to secoiridoids, nuzhenide and GI3, whose assay has been previously reported (Flanagan et al. 2013), the occurrence of cumarins content in Glucevia®, including fraxin, fraxetin, esculin, esculetin, cichoriin, scopolin and fraxidin glucoside was quantified.
On the basis of literature, it is known that Fraxinus extract improves liver metabolism, and reduces fatty liver. Vitamin E and N-acetyl cysteine are antioxidants which help in the improvement of liver function.

When the prior art is taken into consideration, treatment of steatosis is inevitably linked to obesity, insulin resistance and dyslipidemia. Also treatment is bound to administration of antioxidants, probiotics, anti-cytokines, anti-fibrotics and glutathione precursors. However, there is need of combining two or more therapeutic agent to treat NAFLD. The necessity of combining two or more active agents that show synergistic effect, in a single dosage form to treat NAFLD arises from the unavailability of such formulations. Complexity in taking multiple drugs is eliminated by the use of a combined medication in the form of a single dosage form and lead to improved patent compliance. Additive and/or synergistic effect of the combination in treating NAFLD is confirmed through the pharmacological evaluation conducted by the inventors of the application. Therefore, the main object of the present invention is to provide a novel combination composition effective in treating NAFLD.
The present invention is directed to a triple or double combination of Vitamin E, Fraxinus excelsior and/or N-acetyl-L-cysteine (NAC) as active ingredients so as to improve patient compliance to the treatment and thus to achieve treatment success.
The main object of the present invention is to provide such a combination composition in a single unit dosage form. The single unit dosage form of the present invention can be administered by the oral or parenteral route though it is preferably administered by the oral route.
The single unit dosage form of the present invention can be in solid forms such as pill, tablet, capsule, film tablet, orally disintegrated tablet, enterically coated tablet, modified release tablet, prolonged release tablet, delayed release tablet, effervescent powder, effervescent granule, effervescent tablet, sachet, dragee, pastil or in liquid form such as suspension, emulsion, syrup, drop, solution.

In terms of ensuring precise and accurate dose adjustment and having long shelf life, solid dosage forms have appeared more advantageous in scope of the present invention.
The main object of the present invention is to provide such a combination composition in a solid oral dosage form particularly in the form of soft gelatin capsule.
Extract of various parts of the F. excelsior tree may be selected from leaves, bark, fruits, seeds and combinations thereof but more preferably seed/fruit extract.
One or more pharmaceutically acceptable excipients for capsule shell may be selected from encapsulation material, film forming agents, plasticizers, neutralizers, coloring agents, opacifiers and combinations thereof.
Suitable encapsulation materials used for capsule shell according to the present invention may be selected from gelatin, hydroxypropyl methylcellulose, alginates and combinations thereof. In addition to encapsulation materials, suitable film forming materials used for capsule shell according to the present invention may be selected from polyvinyl pyrrolidone, acrylates, zein, shellac and acrylamides and combinations thereof. Suitable plasticizers used for capsule shell used according to the present invention may be selected from water, polyethylene glycol, glycerin, propylene glycol, sorbitol and combinations thereof.
Suitable coloring agents and opacifiers used for capsule shell according to the present invention may be selected from saffron, brilliant blue, tartrazine, sunset yellow, quinoline yellow supra, titanium dioxide and combinations thereof.
Coloring agents added in capsule shell contains alkali metal ions. When alkali metal salts are added in capsule shell, the excess alkali is to be neutralized by treating the capsules with an acid selected from any of the following, namely,

hydrochloric acid, sulphuric acid, nitric acid, phosphoric acid, mono carboxylic acids such as acetic acid, propionic acid, benzoic acid etc., dicarboxylic acids such as oxalic acid, maleic acid, fumaric acid etc. The acids are used in the form of cold dilute aqueous solutions in the concentration range of 3 to 30% depending on the type of acid used. The acid treatment may be carried out after manufacturing and partial drying of the capsules to avoid deformation and/or leakage of the capsule contents.
The composition fill in capsule of the described invention may be formulated as oily solution and/or suspension. According to the present invention, suitable oil used for dissolving and/or dispersing medicaments may be selected from coconut oil, sunflower oil, hydrogenated vegetable oil and combinations thereof. Preferably soyabean oil is used.
The surface active agent used in capsule fill composition as a solublising and/or dispersing aid is selected from among, but is not restricted to substances such as lecithin, polyoxyethylene castor oil derivative such as Cremophor RH 40 (polyoxyl 40 hydrogenated castor oil), Cremophor EL (polyoxyl 35 castor oil, BASF) polyoxyethylene sorbitan fatty acid esters, Gelucire 33/01 (glycerol esters of fatty acids), sodium lauryl sulphate, docusate sodium and the like. Lecithin improves material flow, and imparts some lubrication during filling.
The amount of such surface active agent present in the composition may range from 2.0 to 20.0 percent preferably 4.0 to 15.0 percent by weight with reference to contents filled in capsule.
The suspending agent is selected from among but not restricted to colloidal silicon dioxide, polyvinylpyrrolidone, microcrystalline cellulose, bees wax etc. The amount of such suspending agent present in the composition may range from 0.5 to 20.0 percent preferably 1.0 to 10.0 percent by weight with reference to the content filled in capsules.

Acidic substances present in capsule fill may affect the gelatin shell therefore alkalizers are added to reduce the effect on capsule shell. Therefore alkalizer/alkaline earth metal salts are selected, but not limited to sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, sodium carbonate, sodium bicarbonate, calcium carbonate, magnesium oxide, magnesium carbonate, magnesium hydrogen carbonate, aluminum hydroxide, magnesium hydroxide, magnesium silicate, magnesium aluminate or aluminum magnesium hydroxide, sodium citrate, pharmaceutically acceptable salt of phosphoric acid such as dibasic calcium phosphate or mixtures thereof.
The antioxidant used in capsule incorporating composition of the present invention may be selected from butylated hydroxytoluence, butylated hydroxyl anisole, ascorbic acid, tartaric acid and the like.
The following example is provided to enable one skilled in the art to practice the invention and is merely illustrative of the present invention. The example should not be read as limiting the scope of the present invention.

Example 1:
Vitamin E and Fraxinus excelsior soft gelatin capsule
Each soft gelatin capsule contains:

Sr.
No. Ingredients Quantity
1. Vitamin E
(Equivalent to 268.4 mg of d-α-Tocopherol
derived from vegetable oil) 400 IU
2. Fraxinus excelsior (Seed/Fruit extract) 500 mg
Example 2:
Vitamin E, Fraxinus excelsior and N-acetyl-L-cysteine soft gelatin capsule
Each soft gelatin capsule contains:

Sr. No. Ingredients Quantity
1. Vitamin E
(Equivalent to 268.4 mg of d-α-Tocopherol
derived from vegetable oil) 400 IU
2. Fraxinus excelsior (Seed/Fruit extract) 500 mg
3. N-acetyl-L-cysteine Ph. Eur 300 mg
The above active ingredients were mixed with suitable excipients and the formulation was encapsulated in a soft gelatin capsule according to one of the methods known per se to those skilled in the art.
Further the safety and efficacy of these combinations over individual substances was accessed by pharmacological evaluation in patients with non-alcoholic fatty

liver disease. This study provided evidence that, combinations are clinically relevant pharmacologic approach as an effective and safe medication for the treatment of non alcoholic fatty liver disease in susceptible patients.

We claim,
1. The pharmaceutical composition comprising Vitamin E, Fraxinus excelsior seed extract, N-acetyl-L-cysteine in combination with pharmaceutically acceptable excipients in a single unit dosage form for the prevention or treatment of fatty liver disease.
2. The pharmaceutical composition according to claim 1, wherein the fatty liver disease is non-alcoholic fatty liver disease (NAFLD).
3. The pharmaceutical composition according to claim 1, wherein the composition is formulated as an oral composition.
4. The pharmaceutical composition comprising 400 IU Vitamin E, 500 mg Fraxinus excelsior seed extract and 300 mg N-acetyl-L-cysteine in combination with pharmaceutically acceptable excipients in a single unit dosage form for the prevention or treatment of non-alcoholic fatty liver disease.
5. The pharmaceutical composition comprising 400 IU Vitamin E and 500 mg Fraxinus excelsior seed extract in combination with pharmaceutically acceptable excipients in a single unit dosage form for the prevention or treatment of non-alcoholic fatty liver disease.
6. The oral pharmaceutical composition as claimed in claim 4-5, wherein the combination composition is encapsulated within a soft gelatin capsule.
' 7. The soft gelatin capsule of claim 6, wherein said excipients comprises one or more encapsulation material, film forming agents, plasticizers, neutralizers, coloring agents, opacifiers, suspending agent, surface active agent, solublising agent, alkalizers, antioxidant and combinations thereof.

8. A method for prevention or treatment of non-alcoholic fatty liver disease by administering 400 IU Vitamin E, 500 mg Fraxinus excelsior seed extract and 300 mg N-acetyl-L-cysteine in combination.
9. A method for prevention or treatment of non-alcoholic fatty liver disease by administering 400 IU Vitamin E and 500 mg Fraxinus excelsior seed extract in combination.
10. The pharmaceutical composition according to preceding claims wherein the combination is administered to a mammal for prevention or treatment of non-alcoholic fatty liver disease.