WO2013115636A1 - Composition for the treatment and/or prevention of fatty liver disease - Google Patents

Abstract

There is provided at least one method of treating, preventing, ameliorating, reducing or delaying the onset of fatty liver disease (FLD) using tocotrienol or derivative thereof or a mixture thereof.

Description

COMPOSITION FOR THE TREATMENT AND/OR PREVENTION OF FATTY LIVER

DISEASE

FIELD OF THE INVENTION The present invention relates generally to the field of treating and/or preventing liver diseases. In particular, the present invention may relate to prevention of fatty liver diseases and associated disorders.

BACKGROUND TO THE INVENTION

Fatty liver also known as steatosis, is a disease in which excessive amounts of lipids accumulate in the liver. Fatty liver may develop due to medicine or alcohol use, viral (e.g., Hepatitis C) or bacterial infections or obesity or diabetes. Fatty liver disease (FLD) is a range of liver disorders of increasing severity encompassing from simple accumulation of fat in the hepatocytes (steatosis) to macrovescicular steatosis, periportal and lobular inflammation (steatohepatitis). FLD is usually associated with dyslipidaemia, obesity, insulin resistance, with or without alcohol consumption and type II diabetes and it is also widely accepted that non-alcoholic FLD (NAFLD) represents the liver component of the metabolic syndrome (MetS). FLD patients present a significant increase in cardiovascular (CV) risk, thus linking FLD, MetS and accelerated atherosclerosis together. Fat deposition in the liver or alcoholic or non-alcoholic FLD has emerged to be a liver disorder which is not fully understood both in etiology and significance.

FLD is reported to be the most common cause of chronic liver disease in the United States of America and other western countries with a prevalence rate ranging between 15% to 30% of the adult population and about 10% of this adult population meets the current diagnostic criteria for steatohepatitis (NASH).

Data for Asian populations reported FLD prevalence of 21 % in Shanghai, between 18% and 31.7% in Japan, and 13.5% in Thailand. FLD is also commonly associated with diabetes and it is estimated that 34-74% of diabetic patients have FLD.

There are reported to be over 1 ,000 drugs and chemicals that are capable of causing injury to the liver. The term drug-induced liver disease is used to describe those instances in which an active agent has caused injury to the liver. Drug-induced liver injury may account for as many as 10 percent of hepatitis cases in adults overall, 40 percent of hepatitis cases in adults over fifty years old, and 25 percent of cases of fulminant liver failure. Certain active agents, such as glucocorticoids, synthetic estrogens, amiodarone, tamoxifen and valproic acid, for example, have been associated with fatty liver.

Steatohepatitis (NASH), inflammation of the liver is also related to fat accumulation. Heavy alcohol use can lead to fatty liver and inflammation and is usually referred to as alcoholic hepatitis. Steatohepatitis resembles alcoholic hepatitis, but can occur in people who seldom or never drink alcohol. In this instance, it is often called nonalcoholic steatohepatitis or NASH. Both alcoholic hepatitis and steatohepatitis can lead to scarring, e.g., cirrhosis, and hardening of the liver resulting in serious liver damage. The understanding of fatty liver is thus still an ambiguity and requires further research.

Nutritional assessment of FLD patients revealed low plasma concentration of antioxidants and in particular a lower-than-recommended intake of vitamin E. According to Birringer M. et al. (2002) the metabolic rate of individual forms of vitamin E should markedly affect their bioavailability and bioequivalence in vivo. The most common homologue of vitamin E, alpha-tocopherol, has been studied as a promising agent for amelioration of NASH. However, Bjelakovic G et al. (2011 ) in their review on antioxidant supplements for liver diseases confirmed that convincing evidence that beta-carotene, vitamin A, vitamin C, and vitamin E or their combinations are beneficial for treatment of alcoholic, autoimmune, hepatitis B or hepatitis C virus liver diseases, or liver cirrhosis could not be found. Bjelakovic G ef al. (2008) in another review on antioxidant supplements for prevention of mortality also concluded that use of antioxidant supplements such as Vitamin E is not recommended as a preventive measure against diseases including those associated with the liver.

Accordingly, there is still a need for methods and compositions for an improved and better treatment and/or prevention of the development of fatty liver and conditions stemming from fatty liver, such as NASH, liver inflammation, cirrhosis and liver failure. SUMMARY OF THE INVENTION

The present invention addresses the problems above, and in particular provides at least one method and/or composition that may be used in the prevention of FLD and/or a related disease.

According to a first aspect, the present invention provides at least one method of preventing, or delaying the onset of fatty liver disease (FLD) and/or FLD associated disease(s) in a subject, the method comprising administering a composition comprising at least one tocotrienol, derivative or a mixture thereof to the subject.

According to other aspects, the present invention provides a composition comprising at least one tocotrienol, derivative or a mixture thereof for treating, preventing, ameliorating, reducing or delaying the onset of fatty liver disease (FLD) and/or FLD associated disease(s), and use of the composition comprising at least one tocotrienol, derivative or a mixture thereof for treating, preventing, ameliorating, reducing or delaying the onset of fatty liver disease (FLD) and/or FLD associated disease(s).

As will be apparent from the following description, preferred embodiments of the present invention allow an optimal use of at least one tocotrienol, derivative or a mixture thereof to take advantage of their efficiency and effectiveness in treating, preventing, ameliorating, reducing or delaying the onset of FLD. This and other related advantages will be apparent to skilled persons from the description below.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 are pictures of histological findings of mice. (A) is a picture of histological findings in control mice after hematoxylin-eosin staining; (B) is a picture of histological findings in mice fed High fat high fructose (HFF) diet after hematoxylin-eosin staining; (C) is a picture of histological findings in mice fed HFF and tocotrienols (T3) diet after hematoxylin-eosin staining (magnification x40). Figure 2 is a histogram showing hepatic content of gamma-, alpha-tocotrienols and alpha- tocopherol in mice fed HFF-T3 diet (±SD).

Figure 3 are pictures showing the comparison of histological findings in control (A) [mouse No. 1], (B) HFF diet [mouse No. 5] and (C) HFF-T3 diet [mouse No. 8] after hematoxylin- eosin staining (magnification x4). Arrows indicate lymphomonocytic infiltration.

Figure 4 are pictures showing the comparison of histological findings in control (A) [mouse No. 2], (B) HFF diet [mouse No. 4] and (C) HFF-T3 diet [mouse No. 7] after hematoxylin- eosin staining (magnification x10). Arrows indicate areas of microvesicular steatosis.

Figure 5 are pictures showing the comparison of histological findings in control (A) [mouse No. 3], (B) HFF diet [mouse No. 6] and (C) HFF-T3 diet [mouse No. 9] C after hematoxylin- eosin staining (magnification x40). Arrows in specimen B indicate former areas of fat infiltrates or microvesicular steatosis (white areas) that are absent in specimens A and C.

DETAILED DESCRIPTION OF THE INVENTION

Bibliographic references mentioned in the present specification are for convenience listed in the form of a list of references and added at the end of the examples. The whole content of such bibliographic references is herein incorporated by reference. Definitions

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

As used herein, the term "fatty liver disease," which is also called fatty liver, is referred to a disease leading to liver injury caused by abnormal fat accumulation in liver cells. FLD may arise from a number of sources, including excessive alcohol consumption and metabolic disorders, such as those associated with insulin resistance, obesity, and hypertension. Nonalcoholic fatty liver disease (NAFLD) may also result from metabolic disorders such as, e.g., galactosemia, glycogen storage diseases, homocystinuria, and tyrosemia, as well as dietary conditions such as malnutrition, total parenteral nutrition, starvation, and overnutrition or iron overload. In certain cases, NAFLD is associated with jejunal bypass surgery. Other causes include exposure to certain chemicals such as, e.g., hydrocarbon solvents, and certain medications, such as, e.g., amiodarone, corticosteroids, estrogens (e.g., synthetic estrogens), tamoxifen, maleate, methotrexate, nucleoside analogs, and perhexiline. Acute fatty liver conditions can also arise during pregnancy. Fatty liver disease can be classified into alcoholic liver disease and nonalcoholic fatty liver disease. Diseases falling within the scope of fatty liver disease in the context of the present invention may include but are not limited to the following:

alcoholic liver disease (also called alcoholic liver injury) which is a disease caused by fat accumulation in liver cells as a result of alcohol ingestion. Examples include but are not limited to diseases such as alcoholic simple fatty liver, alcoholic steatohepatitis (ASH), alcoholic hepatic fibrosis, alcoholic cirrhosis and the like. It should be noted that alcoholic steatohepatitis is also called alcoholic fatty hepatitis and includes alcoholic hepatic fibrosis;

- nonalcoholic fatty liver disease which is a disease with fat deposition in the liver, which occurs in patients whose alcohol ingestion is not enough to cause liver injury, except for cases of known etiology, such as viral hepatitis and autoimmune hepatitis. Examples include diseases such as nonalcoholic simple fatty liver, nonalcoholic steatohepatitis (NASH), nonalcoholic hepatic fibrosis, nonalcoholic cirrhosis and the like;

nonalcoholic simple fatty liver which is a disease only with fat deposition in liver cells and/or parenchyma;

nonalcoholic steatohepatitis (NASH) which is a disease with liver fatty change, along with inflammation, liver cell necrosis, ballooning and fibrosis, similarly to the case of alcoholic steatohepatitis, and also including nonalcoholic hepatic fibrosis; nonalcoholic hepatic fibrosis which is a disease with advanced fibrosis in liver tissues, along with excessive production and accumulation of collagen and other

. extracellular matrix components;

nonalcoholic cirrhosis which is a disease with reconstructed hepatic lobule structure as a result of advanced fibrosis;

and the like.

As used herein, the term "pharmaceutically acceptable salt" refers to an acid addition salt or a salt with a base. Specific examples include but are not limited to acid addition salts with mineral acids (e.g., hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid), organic acids (e.g., formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid) or acidic amino acids (e.g., aspartic acid, glutamic acid); salts with inorganic bases (e.g., sodium, potassium, magnesium, calcium, aluminum), organic bases (e.g., methylamine, ethylamine, ethanolamine) or basic amino acids (e.g., lysine, ornithine); as well as ammonium salt, and the like.

The term "comprising" is herein defined to be that where the various components, ingredients, or steps, can be conjointly employed in practicing the present invention. Accordingly, the term "comprising" encompasses the more restrictive terms "consisting essentially of and "consisting of."

As used herein, the term "tocotrienol" encompasses their counterparts bearing unsaturated tails, including, but not limited to, alpha-, beta-, gamma-, and delta- tocotrienols, desmethyl- tocotrienol, didesmethyl-tocotrienol, occurring in sunflower seeds, vegetable oils, barley, brewer's grains, oats, and African violets, their synthetic counterparts, their counterparts having methylated or demethylated chroman rings, and mixtures thereof. The double bonds may be cis or trans or mixtures thereof. In particular, the four tocotrienol isomers comprise a chroman ring, a tail and an active group on the chroman ring of the molecule, which is called the hydroxy group. The chroman ring may have chemical groups which are called methyl groups attached to it. Alpha tocotrienol has all three available sites filled while beta tocotrienol and gamma tocotrienol have two methyl groups but in different positions and delta tocotrienol has only one methyl group as summerised in Table 1 below.

The tocotrienol tail has three double bonds.

The tocotrienol may have the following structure:

Tocotrienols Molecule

Rl R2 R3

alpha- tocopherol cm cm cm

alp a-tocotr iehol

beta -tocopherol

beta-tocotrienol cm H cm

ga m m a-tocoph ero i

gamm a- tocgtr ienol H CH3 CH3

^'delta-tocopherpl_.

delt a-tocotr ienol H H cm

Table 1. Structure of tocotrienol

Homologs of tocotrienols may be chemically synthesized. Experimental details are provided on a new tocotrienol synthesis which provides stereochemical^ pure materials.

The tocotrienols used in any aspect of the present invention include tocotrienol-rich- fractions obtained from natural products as well as the pure compounds. Tocotrienol or tocotrienol-enriched preparations include but are not limited to those containing tocotrienol and, in some cases, tocopherol derivatives, particularly stabilized derivatives. These typically include derivatives related to the phenolic hydroxyl functionality, wherein it is acylated with an organic acid to form an ester. Examples of such stabilized tocotrienols include, but are not limited to, tocotrienol acetate, tocotrienol succinate, and mixtures thereof. Another example of a tocotrienol derivate includes a chromanol nucleus and three double bonds in the hydrocarbon tail. However, the derivatives may also include those involving other reactive groups known to those skilled in the art. Where tocotrienol derivatives are employed, they must be functionally equivalent to tocotrienol. The term "subject" is herein defined as vertebrate, particularly mammal, more particularly human. For purposes of research, the subject may particularly be at least one animal model, e.g., a pig, horse, mouse, rat, cow, dog, cat and the like.

A person skilled in the art will appreciate that the present invention may be practiced without undue experimentation according to the method given herein. The methods, techniques and chemicals are as described in the references given or from protocols in standard biotechnology and molecular biology text books.

According to a first aspect, the present invention provides a method of treating, preventing, ameliorating, reducing or delaying the onset of fatty liver disease (FLD) and/or FLD associated diseases in a subject, the method comprising administering a composition comprising at least one tocotrienol, derivative thereof or a mixture thereof to the subject.

In particular, at least one tocotrienol may have a preventive activity in at least NAFLD in mice, human being and the like. The examples confirm the NAFLD-preventive activity of mixed tocotrienols. In particular, the steatogenic diet fed to mice caused the onset of NAFLD in animals not fed mixed tocotrienols. Tocotrienols have shown NAFLD-preventive activity and evaluation of the hepatic content of tocotrienols univocally related the hepatoprotective efficacy and NAFLD-preventive activity of mixed tocotrienols with the absence of NAFLD, as confirmed by the histology studies in the Examples.

The tocotrienol compound can be produced by known method, such as pressing a natural material, extraction from a natural material, synthesis of the like. The tocotrienol compound may be purified, for example, by column chromatography. Many tocotrienols useful for the practice of the invention are natural products isolated, for example, from wheat germ oil, bran, or palm oil using any method known in the art. For example, high performance liquid chromatography may be used to extract tocotrienols, or tocotrienols may be isolated by alcohol extraction and/or molecular distillation from barley, brewer's grain or oats. Mixed tocotrienols, single isomers, chiral variations and synthetic or semi-synthetic derivatives, in free formulation, inclusion complexes, liposomal formulations, lipophilic and/or hydrophilic matrices, self-emulsifying preparations, controlled release and/or immediate release formulations in solid, liquid or aerosol preparations, for oral, injectable, topical, patch or suppository use may be used in any aspect of the present invention.

In particular, since tocotrienols occur naturally in high amounts in the fruits of the oil palm (Elaeis guineensis), they may be extracted from them for commercial purposes. Tocotrienols may also be extracted from other sources or be produced synthetically de novo.

Tocotrienol or tocotrienol derivatives or mixtures thereof that may be used in any aspect of the present invention may be in the substantial absence of tocopherols wherein the compositions contain essentially no tocopherol.

In particular, the tocotrienol may be a-tocotrienol, β-tocotrienol, y-tocotrienol, δ-tocotrienol, their natural, semi-synthetic or synthetic esters or a mixture thereof. The tocotrienol may be a nicotinate esters of the above tocotrienol isomers, and the like. The tocotrienol compound may be any of d-, 1 or d1 -isomer, and further may be a mixture of two or more of the above tocotrienol compounds. Tocotrienol may be used for its fatty liver preventive and therapeutic activity. The tocotrienol fed to the patient may comprise at least a-tocotrienol, γ-tocotrienol and/or δ- tocotrienol. In particular, the tocotrienol fed to the patient may comprise at least a- tocotrienol and γ-tocotrienol. More in particular, the ratio of α-tocotrienol and γ-tocotrienol may vary depending on the purpose of the tocotrienol fed to the patient. For example, if the tocotrienol is fed for treatment of liver disease, the ratio of α-tocotrienol and γ-tocotrienol may be in the range of 2:1 to 1 :7.

In another example, if the tocotrienol is fed for prevention of liver disease, the ratio of a- tocotrienol and γ-tocotrienol may be in the range of 4:1 to 1 :4. The composition may be a pharmaceutical composition. The pharmaceutical preparation based on the pharmaceutical composition of the present invention can be prepared in a conventional manner by using tocotrienol, tocotrienol derivative or mixtures thereof or a pharmaceutically acceptable salt thereof and a pharmaceutical carrier, a pharmaceutical excipient or other additives commonly used for formulation purposes. The pharmaceutical composition may also include dietary supplements and the like. In particular, the composition may be administered as tablet, or gel, or dragee, or sustained-release formulation, or immediate release formulation or ointment, or injectable formulation or in encapsulated form. In these solid compositions, using tocotrienol, tocotrienol derivative or mixtures thereof or a pharmaceutically acceptable salt thereof may be mixed with at least one inert diluent, for example, lactose, mannitol, glucose, hydroxypropylcellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, magnesium aluminometasilicate or the like. The compositions may also comprise additives in addition to the inert diluent(s), as exemplified by lubricants (e.g., magnesium stearate), disintegrants (e.g., calcium carboxymethyl cellulose), stabilizers, solubilizers and so on, as in the usual cases. Tablets or pills may optionally be coated with sugar coating or a gastric or enteric film, as exemplified by sucrose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose phthalate or the like. Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs, etc., and comprise commonly-used inert diluents such as purified water or ethanol. These compositions may comprise, in addition to the inert diluents, auxiliaries (e.g., wetting agents, suspending agents), sweeteners, flavors, aromatics, and/or antiseptics.

Since vitamin E and their isoforms are in general water insoluble, the compositions referred to herein are in one embodiment prepared in a water soluble form. Thus, the compositions referred to herein are water solubilized by the addition of specific compounds. A water solubilized form of a composition referred to herein can be obtained, for example, by formulating it into a solid dispersion. Other methods of formulating water-5 dispersible or water-soluble tocotrienol forms are disclosed for example in US 5869704.

Injections for parenteral administration comprise sterile aqueous or non-aqueous solutions, suspensions or emulsions. Examples of aqueous solutions or suspensions include injectable distilled water and physiological saline. Examples of non-aqueous solutions or suspensions include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), alcohols (e.g., EtOH), Polysorbate 80, etc. These compositions may further comprise auxiliaries such as antiseptics, wetting agents, emulsifiers, dispersants, stabilizers and/or solubilizers. They are sterilized, for example, by filtration through a bacteria-retaining filter, by incorporation with disinfectants or by irradiation. Alternatively, they may be formulated into sterile solid compositions and reconstituted for use by being dissolved in sterile water or a sterile injectable solvent before use. In general, for oral administration, the daily dosage is desirably about 0.001 to 100 mg/kg, preferably 0.1 to 30 mg/kg, and more preferably 0.1 to 10 mg/kg body weight, given as a single dose or in 2 to 4 divided doses. For intravenous administration, the daily dosage is desirably about 0.0001 to 10 mg/kg body weight, given in one or several doses per day. Likewise, for transmucosal formulations, the daily dosage is about 0.001 to 100 mg/kg body weight, given in one or several doses per day. The dosage may be determined as appropriate for each case in consideration of symptom, age, sex and so on. It should be noted that a pharmaceutical preparation based on the pharmaceutical composition of the present invention can be used in combination with other drugs which are used for treatment of fatty liver disease. For example, drugs which can be used in combination with this pharmaceutical preparation include biguanides (e.g., metformin), thiazolidine derivatives (e.g., pioglitazone hydrochloride), a-glucosidase inhibitors (e.g., voglibose), insulin secretagogues (e.g., nateglinide), vitamins, eicosapentaenoic acid (EPA), betaine, N-acetylcysteine (NAC), fibrate drugs (e.g., bezafibrate), HMG-CoA reductase inhibitors (e.g., atorvastatin), probucol, ursodeoxycholic acid (UDCA), taurine, stronger neo-minophagen C, polyenephosphatidylcholine, phosphatidylcholines, silybin, angiotensin II receptor antagonists (e.g., losartan) or bofutsushosan (oriental herbal medicine), etc. In such combination use, drugs may be administered simultaneously or separately in succession or at desired time intervals. Formulations for simultaneous administration may be in either mixed or separate form.

Formulations for external use include ointments, plasters, creams, jellies, cataplasms, sprays, lotions, eye drops, eye ointments, etc. They comprise commonly-used ointment bases, lotion bases, aqueous or non-aqueous solutions, suspensions, emulsions or the like. Examples of ointment or lotion bases include polyethylene glycol, propylene glycol, white petrolatum, white beeswax, polyoxyethylene hydrogenated castor oil, glycerine monostearate, stearyl alcohol, cetyl alcohol, Lauromacrogol, sorbitan sesquioleate and so on.

Transmucosal formulations such as inhalants or transnasal formulations are used in solid, liquid or semi-solid form and can be prepared in a conventionally known manner. For example, such formulations may be supplemented as appropriate with known excipients and further with pH adjusters, antiseptics, surfactants, lubricants, stabilizers, thickeners and so on. For their administration, an appropriate device for inhalation or insufflation may be used. For example, using a known device (e.g., a metered-dose inhalation device) or a nebulizer, each compound may be administered alone or as a powder of a formulated mixture or as a solution or suspension in combination with a pharmaceutically acceptable carrier. Dry powder inhalators or the like may be for single or multiple administration use, and dry powders or powder-containing capsules may be used in such devices. Alternatively, they may be in the form of pressurized aerosol sprays which use an appropriate propellant, for example, a preferred gas such as chlorofluoroalkane, hydrofluoroalkane or carbon dioxide.

According to a further aspect, the present invention provides a use of at least one tocotrienol, derivative or a mixture thereof for the preparation of a medicament for treatment, prevention, amelioration, reduction or delay of the onset of fatty liver disease (FLD) in a subject.

According to another aspect, the present invention provides a composition comprising at least one tocotrienol, derivative or a mixture thereof for use in treating, preventing, ameliorating, reducing or delaying the onset of fatty liver disease (FLD) in a subject.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention.

A person skilled in the art will appreciate that the present invention may be practised without undue experimentation according to the method given herein. The methods, techniques and chemicals are as described in the references given or from protocols in standard biotechnology and molecular biology text books.

EXAMPLES

Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Sambrook and Russel, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2001).

EXAMPLE 1

Histological findings

The study is a completely randomised interventional study. Animals (Mus musculus or Swiss albino mice, sourced from the animal facilities of Universiti Sains Malaysia (Penang, Malaysia) were randomised into 3 groups of treatment with the first group (Control) fed a standard diet of commercially sourced P702 pellets (Gold Coin, Port Klang, Malaysia), the second group fed high fat-high fructose (HFF) diet comprising 50% P702, 30% clarified butter and 20% fructose and the third group a high-fat-high fructose plus tocotrienols (HFF- T3) diet comprising 50% P702, 30% clarified butter and 20% fructose plus mixed tocotrienols (Tocomin, Carotech, Ipoh, Malaysia). The animals had free access to food and water throughout the length of the experiment. The animals were humanely sacrificed, according to guidelines, for liver harvesting at week 26 from initiation of the study. Liver grafts from HFF-T3 and control animals were retained for chromatographic determination of hepatic tocotrienols content. Tissue quantification of tocotrienols correlates with the effectiveness of the treatment.

The control group showed normal histology (Figure 1A), whereas in the HFF group hepatic lesions were evident (Figure 1 B). The observed lesions, present in all specimens from each mouse, consisted of moderate lymphomonocytic infiltrations with steatosis. The group fed HFF-T3 diet, did not display any sign of hepatic lesion and conformed to normal morphology and histology (Figure 1C).

Tocotrienols hepatic content

In liver specimens from the control group none of the tocotrienols were above the limit of quantification. On the other hand, in the HFF-T3-fed mice specimens, the average gamma- tocotrienol content was 1044.2ng/g (±627.5ng/g), alpha-tocotrienol measured 150.6ng/g (±84.7ng/g). However, it was not possible to quantify delta-tocotrienol due to the presence of an interfering peak at analogous retention time. In addition, alpha-tocopherol levels were measured and stood at 11.0ng/g (±17.0ng/g) for the HFF-T3 group and below quantification in the control group. In figure 2, average concentrations of tocotrienols and alpha-tocopherol for HFF-T3-fed mice are shown. The alpha-tocopherol present in the mixed tocotrienols preparation used in the study was far below the dosage employed in other studies investigating the hepatoprotective activity of alpha-tocopherol alone or in combination with other agents non comprising tocotrienols. Thus, proving that the hepatoprotective activity was solely due to the use of at least one tocotrienol, derivative or a mixture thereof.

The group of animals fed steatogenic diets (HFF) developed fatty liver, as confirmed by histological evaluation. The group that was fed the same steatogenic diet fortified with T3 (HFF-T3) did not develop fatty liver, thus proving the fatty liver preventive effect of T3. The experiment was repeated including alcohol to the animal diet and proved the effectiveness of T3 in prevent alcoholic related fatty liver. Not all the animals on HFF were sacrificed at the end of the first part of the experiment, a subgroup of them was retained to test the curative effects of T3. Since the animals had similar genetic pool and were fed identical HFF diet, the consideration of them having developed fatty liver was statistically justified. This subgroup of animals was changed from their original HFF diet to HFF-T3 diet for a time analogous to the original HFF diet treatment. They were finally sacrificed and were found to be free and cured from fatty liver, thus confirming the therapeutic effects of T3. The experiment was repeated including alcohol to the animal diet and proved the effectiveness of T3 in prevent alcoholic and nonalcoholic related fatty liver.

Example 2 Study Design

The study, designed as a parallel randomised interventional study, was approved by the Animal Ethics Committee of Universiti Sains Malaysia (AECUSM). Nine animals were randomised into 3 groups of treatment with the first group (Control) fed a standard diet, the second group fed high fat-high fructose (HFF) diet and the third group a high-fat-high fructose plus tocotrienols (HFF-T3) diet. The animals had free access to food and water throughout the length of the experiment. The animals were humanely sacrificed using carbon dioxide inhalation, according to guidelines, for liver harvesting at week 26 from initiation of the study. Liver grafts from HFF-T3 and control animals were retained for chromatographic determination of hepatic tocotrienols content.

Animals

4-week old, male Swiss albino mice (Mus musculus) were received after weaning, accustomed to the new environment for one week on a standard pellets diet. The animals were subsequently randomised to their respective group of treatment. The mice, housed in single cages to avoid competition for their assigned feed, were kept on natural light/dark cycle (that in the equatorial region approximates to 12 hours each) at room temperature (24±2D). Body-weight of the animals was taken weekly.

Animal Diet

The animals assigned to the control group received throughout the study duration a standard diet consisting in P702 pellets (Gold Coin, Port Klang, Malaysia). The HFF diet group received a in-house developed and extruded feed consisting of a mixture of standard P702 pellets powder, dairy fat (Kee Wee, Penang, Malaysia) and fructose (HMBG Chemicals, Hamburg, Germany). Similarly, the HFF-T3 feed was prepared from the HFF ingredients and enriched with tocotrienols (Tocomin50%, Carotec, Ipoh, Malaysia). The in- house feed was prepared weekly and stored at -20□ until dispensed. Details of the composition of the developed feed is given in table 2 below

The amount of tocotrienols to be fed to the animals was extrapolated according to the human-mice equivalent dose equation based on Body Surface Area (BSA) (Reagan- Shaw et al, 2007):

Human Equivalent Dose (mg/kg) = Mice dose (mg/kg) * Mice Km I Human Km

Where the Km factor is equal to 3 for Mice Km and equal to 37 for Human Km.

The amount of tocotrienols in the HFF-T3 pellets was proportionally increased with the weight-gain of the animals throughout the study period. The mixed tocotrienols approximately corresponding to a daily dose of 400 mg for an adult human of 60 kg, is equivalent to 6.7 mg/kg. Thus, based on the above equation, each animal fed the HFF-T3 diet received approximately 82.2 mg/kg of mixed tocotrienols. The developed steatogenic- tocotreinol free diet proved to be a valid tool for onset of NAFLD in mice, causing hepatic changes in all animals fed such diet.

Histological Evaluation

After the death of the animals was ascertained, the liver was harvested following a midline laparotomy, rinsed with saline and immediately placed in 10% formaldehyde for 2 weeks. Subsequently, the specimens underwent tissue processing, paraffin embedding and were microtomed in 3 pm-thick individual sections. Evaluation of the pathological changes in the liver was obtained using a standard hematoxylin and eosin staining procedure. The histopathological assessment was confirmed by an experienced veterinary (NAK), blinded to the treatment assigned to the animals.

Tocotrienols Hepatic Content Evaluation Liver grafts from control and HFF-T3 animals, after being rinsed with saline, were stored at -20° C till analysis for evaluation of the hepatic content of tocotrienols. Them thawed portions were homogenised using mortar and pestle. The average content of tocotrienols and alpha-tocopherol were normalised per 1 g of specimen. The analytical method employed for the determination of hepatic content of tocotrienols was developed and validated by Cheng (2005). Briefly, tocotrienol was extracted from homogenised liver grafts weighing 250 pg each by addition of 1 ml of ethanol, followed by 4 ml of extraction solvent consisting of hexane:ethyl acetate (95:5 v/v). The prepared samples were first vortexed for 2 min then centrifuged at 3500 rpm for 10 min. A 50 μΙ aliquot of supernatant was injected into a Waters (Milford, MA, USA) high-performance liquid chromatography (HPLC) system, consisting of autosampler 717 Plus, separation module 600Control and fluorescent detector 2475. The detector excitation wavelength was set 296 nm and the emission wavelength was set 330 nm. An Allsphere (Alltech, Deerfield, IL, USA) silica column (250x4.6 mm, 5 μ), fitted with a silica-filled guard column, was employed for the normal phase analysis. The mobile phase used throughout the chromatographic evaluation consisted of hexane:tetrahydrofuran (100:4 v/v), at a flow rate of 2 ml/min. Hexane, tetrahydrofuran and ethyl acetate were sourced from Merck (Darmstadt, Germany). All the chemicals and reagents employed were of analytical or HPLC grade.

The standard solutions of tocotrienols were freshly prepared in hexane by serial dilution of Tocomin® 50% to obtain concentrations ranging from 6140.0 ng/ml to 1.5 ng/ml for alpha- tocotrienol, from 10300.0 ng/ml to 2.5 ng/ml for gamma-tocotrienol and from 2640.0 ng/ml to 0.6 ng/ml for delta-tocotrienol and from 1313.8 ng/ml to 0.6 ng/ml for alpha-tocopherol. The limit of quantification (LOQ) was 1.5 ng/ml, 2.5 ng/ml and 0.6 ng/ml for alpha-, gamma- and delta-tocotrienol respectively and 0.6 ng/ml for alpha-tocopherol. The standard curve (n=6) was found to be linear over the concentration range considered, with a Pearson's correlation coefficient of 0.9998, 0.9998 and 0.9999 for alpha-, gamma- and delta- tocotrienol and 0.9987 for alphatocopherol. For the range of concentrations considered, the recovery ranged between 93.7% and 101.3% for alpha-tocotrienol, between 94.5% and 101.0% for gamma- tocotrienol, between 95.1 % and 99.4% for delta-tocotrienol. For alpha- tocopherol the recovery range was between 80.7% and 97.9%. The coefficient of variation, indicating the precision of the assay, was in the range 2.0%-6.7%, 1.2%-4.3% and 0.5%- 4.9% for alpha-, gamma- and delta-tocotrienol respectively. For alpha-tocopherol the coefficient of variation ranged between 1.6% and 7.9%.

On the other hand, the within-day precision of the assay, which is shown by the coefficient of variation, ranged from 1.4% to 11.4% for alpha-tocotrienol, from 1.4% to 5.1 % for gamma-tocotrienol, from 0.5% to 11.7% for delta-tocotrienol and from 1.5% to 6.3% for alpha-tocopherol. The accuracy range was 94.1 %-1 11.7%, 92.0%-107.2%, 94.2%-100.4% and 95.9%-105.5% for alpha-, gamma- and deltatocotrienol and alpha-tocopherol, respectively.

The between-day accuracy of the assay was in the range 97.2%-108.8%, 89.5-100.7%, 96.2%-105.4% and 96.6%-102.9% for alpha-, gamma- and deltatocotrienol and alpha- tocopherol, respectively. Meanwhile, the precision was in the range 1.6%-11.4%, 1.2%- 7.8%, 0.2%-3.9% and 1.4%-3.1 % for alpha-, gamma- and delta-tocotrienol and alpha- tocopherol, respectively.

Results

Overall, average body-weight of the mice (n=9) was 17.1 g ± 3.0 g (range 11.0-21. Og) when received (Week-0) from the Animal House Unit of Universiti Sains Malaysia, 21.4 g ± 3.5 g (range 16.0-26.0 g) at initiation of the experiment (Week-1) and progressively gained weight up to 44.0 g ± 4.9 g (range 36.5-53.5 g) in conjunction with the end of the study (Week-26). Liver weight was approximately 5% of the body-weight in all groups. Shown in table 3 details of the body-and liver-weights. The daily intake of feed proportionally increased to approximately 5 g per animal starting from Week-6 of the experiment.

Table 3: Average values of mice weight, liver weight (after harvesting at 26-week) and liver to body mass ratio by group ±SD (C=control diet; HFF=high fat-fructose diet; HFF-T3=high fat-fructose diet enriched with tocotrienols)

It was observed that animals in the control group were, as expected, of normal body size, whereas the HFF and HFF-T3 were slightly obese. During liver harvesting, abdominal fat accumulation in both HFF and HFF-T3 treated animals was noticeable, on the other hand in the control group there were not sign of abnormal abdominal fat presence. Macroscopically, liver specimens from the HFF diet group appeared to be darker than both control and HFF-T3 groups.

Histological Findings

The control group showed normal histology, whereas in the HFF group hepatic lesions were evident. The observed lesions, present in all specimens from each mouse, consisted of moderate lymphomonocytic infiltration with steatosis. The group fed HFF-T3 diet, did not display any sign of hepatic lesion and conformed to normal morphology and histology. Sample images from all mice are given in figure 5. The present was a preliminary study on the NAFLD-preventive effects of tocotrienols. The histopathological findings were similar for all animals of each of the 3 groups of treatment, as assessed by a blinded veterinary. All mice fed a standard pellets diet (controls) did not develop NAFLD. All mice fed the steatogenic diet (HFF diet) developed NAFLD. All mice fed the tocotrienols-enriched diet (HFFT3) did not develop NAFLD. Thus, considering the consistency of the findings amongst all animals of each group of treatment, the number of nine mice sacrificed was reputed adequate for such preliminary investigation.

Steatosis degree was found to be in agreement with other works present in diet related onset literature (Demori et al, 2006; Yamaguchi et al, 2007; Rinella et al, 2008; Akagiri et al, 2008). The histology findings, supported by analytical evaluation of the tocotrienol content, in the HFF-T3 group showed the effectiveness of tocotrienols in preventing NAFLD onset in all mice fed such diet.

Tocotrienols Hepatic Content

In liver specimens from the control group none of the tocotrienols were above the limit of quantification. On the other hand, in the HFF-T3-fed mice specimens, the average gamma- tocotrienol content was 1044.2 ng/g (±627.5 ng/g), alpha-tocotrienol measured 150.6 ng/g (±84.7 ng/g). However, it was not possible to quantify deltatocotrienol due to the presence of an interfering peak at analogous retention time. In addition, alpha-tocopherol levels were measured and stood at 11.0 ng/g (±17.0 ng/g) for the HFF-T3 group and below quantification in the control group. In figure 2, average concentrations of tocotrienols and alpha-tocopherol for HFF-T3-fed mice are shown. The results of the present study showed erratic bioavailability of alpha- and gamma-tocotrienol, also seen in humans.

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Claims

Use of at least one tocotrienol derivative or a mixture thereof for the preparation of a medicament for prevention of onset of fatty liver disease (FLD) in a subject.

The use according to claim 1 , wherein the FLD is at least one disease selected from the group consisting of alcoholic FLD, non alcoholic FLD, steatohepatitis, alcoholic steatohepatitis, non-alcoholic steatohepatitis (NASH), liver inflammation, cirrhosis, and liver failure.

The use according to either claim 1 or 2, wherein the tocotrienol is a-tocotrienol, β- tocotrienol, γ-tocotrienol, δ-tocotrienol and/or their natural, semi-synthetic or synthetic esters.

The use according to any one of the preceding claims wherein the tocotrienol is σ- tocotrienol, γ-tocotrienol or a combination of both.

5. The use according to any one of the preceding claims, wherein the subject is a mammal.

6. The use according to claim 5, wherein the mammal is selected from the group consisting of human, pig, horse, mouse, rat, cow, dog and cat. 7. The use according to any one of the preceding claims, wherein the medicament is in a form selected from the group consisting of a tablet, gel, dragee, sustained-release formulation, immediate release formulation ointment, injectable formulation and in encapsulated form.

A method of preventing onset of fatty liver disease (FLD) and/or FLD associated diseases in a subject, the method comprising administering a composition comprising at least one tocotrienol, derivative or a mixture thereof to the subject.

9. The method according to claim 8, wherein the FLD is at least one disease selected from the group consisting of alcoholic FLD, non alcoholic FLD, steatohepatitis, alcoholic steatohepatitis, non-alcoholic steatohepatitis (NASH), liver inflammation, liver fibrosis, cirrhosis and liver failure.

10. The method according to either claim 8 or 9, wherein the tocotrienol is a-tocotrienol, β-tocotrienol, γ-tocotrienol, δ-tocotrienol and/or their natural, semi-synthetic or synthetic esters.

11. The method according to any one of claims 8 to 10, wherein the tocotrienol is a- tocotrienol, γ-tocotrienol or a combination of both.

12. The method according to any one of claims 8 to 11 , wherein the subject is a mammal.

13. The method according to claim 12, wherein the mammal is selected from the group consisting of human, pig, horse, mouse, rat, cow, dog and cat.

14. The method according to any one of claims 8 to 13, wherein the composition is in a form selected from the group consisting of tablet, gel, dragee, sustained-release formulation, immediate release formulation ointment, injectable formulation and in encapsulated form.

15. The method according to any one of claims 8 to 14, wherein the composition is administered orally, intradermally, subcutaneously or intraperitoneally. 16. A composition comprising at least one tocotrienol, derivative or a mixture thereof for use in preventing the onset of fatty liver disease (FLD) in a subject.

17. The composition according to claim 16, wherein the FLD is at least one disease selected from the group consisting of alcoholic FLD, non alcoholic FLD, steatohepatitis, alcoholic steatohepatitis, non-alcoholic steatohepatitis (NASH), liver inflammation, liver fibrosis, cirrhosis, and liver failure.

18. The composition according to either claim 16 of 17, wherein the tocotrienol is a- tocotrienol, β-tocotrienol, v-tocotrienol, δ-tocotrienol and/or their natural, semi- synthetic or synthetic esters.

19. The composition according to any one of claims 16 to 18, wherein the tocotrienol is a-tocotrienol, γ-tocotrienol or a combination of both.

20. The composition according to any one of claims 16 to 19, wherein the subject is a mammal.

21. The composition according to any one of claims 16 to 20, wherein the composition is in a form selected from the group consisting of a tablet, gel, dragee, sustained- release formulation, immediate release formulation ointment, injectable formulation and in encapsulated form.

22. The composition according to any one of claims 16 to 21 , wherein the composition is for oral, intradermal, subcutaneous or intraperitoneal administration.