WO2020259493A1

WO2020259493A1 - Compositions and methods for the prevention or treatment, or dietary management of nafld

Info

Publication number: WO2020259493A1
Application number: PCT/CN2020/097698
Authority: WO
Inventors: Kai Lin EK; Yan Qin; Dietrich Rein; Lihua Song
Original assignee: Basf Se; Basf (China) Company Limited
Priority date: 2019-06-24
Filing date: 2020-06-23
Publication date: 2020-12-30

Abstract

Provided herein are compositions and methods for the treatment or dietary management of NAFLD. Particularly, it relates to combinations comprising a first composition comprising a sterol, a stanol, a sterol ester, a stanol ester and/or another sterol derivative and together or separately a second composition comprising a n-3 polyunsaturated fatty acid (n-3 PUFA), a monoacylglyceride of n-3 PUFA, a diacylglyceride of n-3 PUFA, a triacylglyceride of n-3 PUFA and/or another derivative of n-3 PUFA, and methods of using them.

Description

Compositions and methods for the prevention or treatment, or dietary management of NAFLD

Field of the Invention

The present invention relates to compositions and methods for the prevention and/or treatment or the dietary management of NAFLD. Particularly, the present invention relates to combinations comprising a first composition comprising a sterol, a stanol, a sterol ester, a stanol ester and/or another sterol derivative and together or separately a second composition comprising a n-3 polyunsaturated fatty acid (n-3 PUFA) , monoacylglycerides, diacylglycerides, triacylglycerides and/or another derivative thereof, and methods of using them.

Background of the Invention

Non-alcoholic fatty liver disease (NAFLD) is the presence of hepatic steatosis (>5%liver fat assessed by imaging modalities or >5%of cells containing visible lipid droplets from histology) that is not related to significant alcohol consumption, hereditary disorders, viral infection or steatogenic medication. Early NAFLD is typically reversible, but can develop in some 30 %of cases into non-alcoholic steatohepatitis (NASH) , presenting as hepatic steatosis with inflammation, ballooning and evidence of hepatocellular injury with or without fibrosis. In Western countries, NAFLD is the leading cause of liver disease. NAFLD is estimated to affect 20-30%of the general population.

Despite the increasing prevalence of NAFLD and its associated morbidity and mortality, there is currently no approved drug therapy for its treatment. Lifestyle modification consisting of diet, exercise, and weight loss has been advocated to treat patients with NAFLD. However, such lifestyle changes are typically difficult to sustain in the long term, creating a significant unmet need for this condition.

According to the Practice Guidance from the American Association for the Study of Liver Diseases (Chalasani et al., Hepatology Vol. 67 No. 1 (2018) , 328-357) an earlier review of the literature found evidence for supporting the use of omega-3 fatty acids in NAFLD patients. However, two later reported studies failed to show convincing therapeutic benefit for omega-3 fatty acids in patients with NAFLD or NASH.

Tobin et al., Nutrients 10 (2018) , 1126 describe a clinical trial (CONDIN study) for evaluating a high concentrate of omega-3 fatty acids in NAFLD patients. Both the treatment and placebo group patients showed a decrease in liver fat percentage. There was no statistically significant difference in the decrease in liver fat between the two groups. However, a post hoc analysis of the data revealed liver fat reduction in subgroups with a high fatty liver index (FLI) score.

Song et al., Scientific Reports 7 (2017) , 41604 describes a rat animal model of NAFLD wherein the rat animals were administered a diet enriched in saturated fat (lard) and cholesterol. On the basis of the results obtained for the administration of a phytosterol ester in this animal model the authors speculate that phytosterol esters may be useful in the prevention and progression of NAFLD in human patients. However, no clinical data have been described.

In view thereof, there is a need for the provision of compositions and methods suitable for the prevention and/or treatment and/or the dietary management of NAFLD in human patients.

Summary of the Invention

The technical problem underlying the invention is solved by the provision of the subject-matter as defined in the claims.

According to a first aspect is provided a combination comprising a first composition comprising a sterol, a stanol, a sterol ester, a stanol ester and/or another sterol derivative, and together or separately a second composition comprising a n-3 polyunsaturated fatty acid (n-3 PUFA) , a monoacylglyceride of n-3 PUFA, a diacylglyceride of n-3 PUFA, a triacylglyceride of n-3 PUFA, and/or another derivative of a n-3 PUFA for use in the prevention and/or treatment of a human patient suffering from non-alcoholic fatty liver disease (NAFLD) .

According to a second aspect is provided a method for the treatment of a human patient suffering from NAFLD comprising administering to said patient an effective amount of the first composition according to the invention and an effective amount of the second composition according to the invention.

According to a third aspect is provided a kit for pharmaceutical use comprising a first composition comprising campesterol or an ester thereof, stigmasterol or an ester thereof and/or β-sitosterol, an ester or another derivative thereof, and a second composition comprising EPA or a monoacylglyceride, diacylglyceride and/or triacylglyceride thereof or another derivative thereof and/or DHA or a monoacylglyceride, diacylglyceride and/or triacylglyceride thereof or another derivative thereof.

According to a fourth aspect is provided a food supplement for the dietary management of a human patient suffering from NAFLD comprising a first composition comprising campesterol or an ester thereof or another derivative thereof, stigmasterol or an ester thereof or another derivative thereof and/or β-sitosterol or an ester thereof or another derivative thereof, and a second composition comprising EPA or a monoacylglyceride, diacylglyceride and/or triacylglyceride thereof or another derivative thereof and/or DHA or a monoacylglyceride, diacylglyceride and/or triacylglyceride thereof or another derivative thereof.

According to a fifth aspect is provided the use of the first composition according to the invention and the second composition according to the invention in a food supplement for the dietary management of NAFLD.

The inventors have conducted a placebo-controlled clinical trial in NAFLD patients, wherein the co-administration of a first composition comprising a phytosterol ester and a second composition comprising a triglyceride having at least one n-3 PUFA moiety has been assessed in comparison to the administration of the first and second composition, respectively, alone. Surprisingly, as outlined in the examples it has been found that the combination provided a stronger liver fat reduction compared to the administration of the single compounds alone. The effect was synergistic.

Brief Description of the Drawings

Figure 1. The changes in the TG, TC, LDL-C and HDL-C levels of each group after 12 weeks of intervention

A-D. The lipid profiles of all participants in each group.

E-H. The lipid profile of hyperlipidemic participants in each group.

Figure 2. Comparison of patients number with abnormal ALT and GGT before and after intervention

A-D. Patients number with abnormal ALT before and after intervention.

E-H. Patients number with abnormal GGT before and after intervention.

Figure 3. The reduction in the TGF-β (A) and TNF-α (B) levels of the study and control groups

Figure 4. The concentration and increase in the serum DHA and EPA levels of the study and control groups (##means P<0.01) .

Detailed Description of the Invention

Where the term “comprise” or “comprising” is used in the present description and claims, it does not exclude other elements or steps. For the purpose of the present invention, the term “consisting of” is considered to be an optional embodiment of the term “comprising of” . If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which optionally consists only of these embodiments.

Where an indefinite or a definite article is used when referring to a singular noun e.g. “a” or “an” , “the” , this includes a plural form of that noun unless specifically stated.

Vice versa, when the plural form of a noun is used it refers also to the singular form.

Furthermore, the terms first, second, third or (a) , (b) , (c) and the like in the description and in the claims are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

In the context of the present invention any numerical value indicated is typically associated with an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. As used herein, the deviation from the indicated numerical value is in the range of ± 10%, and preferably of ± 5%. The aforementioned deviation from the indicated numerical interval of ± 10%, and preferably of ± 5%is also indicated by the terms “about” and “approximately” used herein with respect to a numerical value.

According to a first aspect is provided a combination comprising a first composition comprising a sterol, a stanol, a sterol ester and/or a stanol ester and together or separately a second composition comprising a n-3 polyunsaturated fatty acid (n-3 PUFA) , a monoacylglyceride of n-3 PUFA, a diacylglyceride of n-3 PUFA, a triacylglyceride of n-3 PUFA and/or another derivative of n-3 PUFA for use in the prevention and/or treatment of a human patient suffering from non-alcoholic fatty liver disease (NAFLD) .

Herein the term “together” means that the first and the second composition are in the same pharmaceutical formulation. This also includes the possibility that the active ingredients of the first composition and the second composition are separated from each other in different compartments or layers of the pharmaceutical formulation.

“Sterols” and their hydrogenated derivatives, the “stanols” , may be derived from plants, animals or fungi. It may be in the form of an extract or in isolated form. The sterol or stanol may alternatively be synthesized. Preferred “sterols” and “stanols” are “phytosterols” (i.e. sterols derived from plants and their hydrogenated derivatives, the phytostanols) such as campesterol, stigmasterol, sitosterol, campestanol, stigmastanol and/or sitostanol.

“Sterol esters” and “stanol” esters may be derived from the esterification of the above defined sterols and stanols. Preferably, phytosterols and/or phytostanols, particularly preferred phytosterols, are esterified with carbonic acids, preferably fatty acids; such esters are the result of the esterification of a hydroxyl group of a sterol or stanol with an acid, preferably a fatty acid. Such fatty acids can be in principle all fatty acids of natural and non-natural origin, preferably of natural origin or obtained from natural origin; esters could be obtained from single or mixed, preferably mixed, fatty acids, said fatty acids having carbon chain lengths of C2 to C24, preferably C4 to C18, particularly C6 to C12, or particularly C16 to C18, such as C3, C4, C5, C6, C7, C8, C9, C120, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, with the even-numbered chain lengths being preferred (meaning C2, C4, C6, C8, C10, C12 etc. up to C24) , and the fatty acids typically being a mixture of more than one chain length, such as mixtures of C4, C6, C8 and C10 at all possible ratios of those chains lengths to each other, preferably mixtures of chain lengths C6 to C10 such as a mixture of C6/C8/C10 and C6/C8 and fatty acid mixtures very rich in C8/C10-fatty acids, or such fatty acid mixtures of C16/C18, polyunsaturated lipids (such as conjugated linoleic acids, docosahexaenoic acid eicosapentaenoic acid) . In another embodiment the fatty acids are the ones being comprised in vegetable oils, like rape oil, soybean oil, olive oil, palm oil etc., or in tall oil and they reflect the fatty acid composition of the natural source as in the crude oil or respective refined oil. Preferred, however, are the fatty acid mixtures of soybean oil, rape oil, and tall oil.

Preferred, however, are fatty acid mixtures containing at least one of the substances selected from a) polyunsaturated fatty acids (also known as “PUFA”s) and conjugated fatty acids (e.g. conjugated linolenic acids) , pinolenic acid and the like. Preferred substances are polyunsaturated fatty acids (PUFAs) , particularly preferred are the so-called omega-3 fatty acids, omega-6 fatty acids and omega-9 fatty acids; b) Monounsaturated fats containing mono-unsaturated fatty acids such as oleic acid, palmitoleic acid and the like; and/or c) Saturated fats containing saturated fatty acids such as propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, Nnonadecylic acid, arachidic acid, heneicosylic acid, behenic acid, tricosylic acid, lLignoceric acid, pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid, nonacosylic acid, melissic acid, henatriacontylic acid, lacceroic acid, psyllic acid, geddic acid, ceroplastic acid and/or hexatriacontylic acid.

Particularly preferred the at least one substance is selected from a) and/or b) above and very particularly preferred at least one substance selected from a) as defined above; in another embodiment very particularly preferred the at least one substance is selected from b) above . Preferably, this at least one substance selected from those as disclosed in paragraphs a) , b) and/or c) above is present in an amount based on the total fatty acid ester content within the sterol ester and/or stanol ester or their mixtures of at least 10 mol percent, preferably at least 20 mole percent, particularly preferably in at least 40 mole percent and very particularly preferably in at least 50 mole percent such as 60, 70, 80, 90 or even 100 mole percent, including all ratios in between those numbers, such as for example 21, 22, 23, 24, 25, 26, 27, 28 and 29, and 31, 32 etc., 41, 42 etc., and 51, 52 etc. and so on.

“another sterol derivative” herein includes phytosterol esters with aromatic or heterocyclic carboxylic acids. The phytosterol is condensed with the aromatic or heterocyclic carboxylic acid under suitable reaction conditions. The aromatic unit may be derived from benzene or a substituted benzene ring. The heterocyclic unit may be derived from pyridine, quinolone, isoquinoline, indole, imidazole, pyrrole, furan, thiophene or substituted variants thereof. The term also includes phytosterol ethers with aromatic or heterocyclic acids. The ether may be prepared by the classical method of ether formation. The term also includes salts of sterol esters and sterol ethers. Alkali salts and/or earth alkali salts are preferred. More preferably, sodium salts and potassium salts. While the weight percentages as used in the following refer to the free form, the composition may contain the free form and/or the ester form. In the latter case, the weight percentage refers to the portion of the phytosterol moiety and/or the phytostanol moiety contained in the ester.

In one embodiment, the first composition comprises at least 30%by weight, preferably at least 40 %, 45 %, 55 %, 65 %, 75 %, 85%or 95 %by weight, of campesterol (determined as free sterols in the sterols profile) , stigmasterol (determined as free sterols in the sterols profile) and/or β-sitosterol (determined as free sterols in the sterols profile) . In one embodiment, the first composition comprises essentially no campesterol, or ester thereof. In another embodiment, the first composition comprises essentially no stigmasterol or ester thereof. Herein, the term “essentially no” means less than 1 %by weight of the composition. In another embodiment the composition comprises at least 60%of β-sitosterol (determined as free sterols in the sterols profile) .

In a further embodiment the first composition comprises essentially no β-sitosterol or ester thereof.

In some embodiments, the weight ratio of campesterol ester: β-sitosterol ester ranges from about 1: 10 to about 10: 1, from about 1: 8 to about 8: 1, from about 1: 6 to about 6: 1, or from about 1: 2 to about 2: 1.

The first composition may have the following composition:

a) a total content of 50 to 90 %sterol ester;

b) 10 %to 40 %campesterol (determined as free sterols in the sterols profile) ;

c) 1 %to 5 %campestanol (determined as free stanols in the sterols profile) ;

d) 10 %to 30 %stigmasterol (determined as free sterols in the sterols profile) ;

e) 10 %to 80 %β-sitosterol (determined as free sterols in the sterols profile) ;

f) 2 %to 15 %β-sitostanol (determined as free stanols in the sterols profile) .

In particular, the first composition is the commercially available product

67WDP from the company BASF.

67WDP is a powder comprising plant sterol and plant stanol fatty acid esters. The carrier system consists of a mixture of sodium caseinate and glucose syrup. Mixed tocopherols and ascorbate are added as antioxidants. 100 g

67WDP comprises 43 g sterols. The sterol composition (on the basis of free sterols) is 0.0-6.0 %brassicasterol, 20.0-29.0%campesterol, 0.0-6.0%campestanol, 12.0-23.0%stigmasterol, 42.0-55.0%β-sitosterol, 0.0-2.5%sitostanol, 0.0-4.0%D5-avenasterol, 0.0-2.0%D7-stigmastenol, 0.0-2.0%D7-avenasterol and 0.0-5.0%other sterols. Alternatively, the commercial product

95 (from BASF) may be used.

95 has a total sterols content of about 59%further including mixed tocopherols and ascorbylpalmitate as antioxidants.

“n-3 PUFA” , also called “omega-3 fatty acids” are polyunsaturated fatty acid with at least 18 carbons, preferably 18 to 22 carbons, and 3 to 6 carbon-carbon double bonds, wherein at least one double bond is in the omega-3 position; preferred omega-3-fatty acids are EPA and DHA; “omega-6 fatty acids” are polyunsaturated fatty acid with at least 18 carbons, preferably 18 to 22 carbons, and 3 to 6 carbon-carbon double bonds, wherein at least one double bond is in the omega-6 position.

In an embodiment the n-3 PUFA is selected from the group consisting of hexadecatrienoic acid (HTA) , α-linolenic acid (ALA) , stearidonic acid (SDA) , eicosatrienoic acid (ETE) , eicosatetraenoic acid (ETA) , eicosapentaenoic acid (EPA) , heneicosapentaenoic acid (HPA) , docosapentaenoic acid (DPA) , docosahexaenoic acid (DHA) , tetracosapentaenoic acid, tetracosahexaenoic acid (nisinic acid) , preferably the n-3 PUFA is EPA or DHA, or a mixture thereof.

In a preferred embodiment in addition to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) , other polyunsaturated fatty acids, particularly omega-3 fatty acids, may be present in the composition. In one embodiment, the composition further comprises at least one fatty acid other than EPA and DHA. The at least one other fatty acid is, for example, selected from the group consisting of from α-linolenic acid (ALA) , heneicosapentaenoic acid (HPA) , docosapentaenoic acid (DPA) , eicosatetraenoic acid (ETA) , eicosatrienoic acid (ETE) , stearidonic acid (SDA) , hexadecatrienoic acid (HTA) , tetracosapentaenoic, tetracosahexaenoic acid, and mixtures thereof.

“A monoacylglyceride of n-3 PUFA, a diacylglyceride of n-3 PUFA and/or a triacylglyceride of n-3 PUFA” are included as compounds for the second composition. The diacylglyceride and/or the triacylglyceride form of n-3 PUFA have one, two or three n-3 PUFA moieties. The two or three n-3 PUFA moieties may be derived from the same n-3 PUFA or may be from a mixture of different fatty acids including the n-3 PUFA. The monoacylglyceride of n-3 PUFA, a diacylglyceride, a triacylglyceride and/or another derivative of n-3 PUFA of the second composition may be artificially synthesized or may originate from an oil, particularly from a marine oil, and most particularly from fish oil, but may also be derived from algae oil, plant-based oil, or microbial oil. As used herein, the term "marine oils" includes oil from fish, shellfish (crustaceans) , and sea mammals. Non-limiting examples of fish oils that can be used as raw oil to manufacture the composition of the invention are, for example, menhaden oil, cod liver oil, herring oil, capelin oil, sardine oil, anchovy oil, salmon oil, mackerel oil, tuna oil, seal oil, and krill oil. The fish oils mentioned above may be recovered from fish organs, e.g. cod liver oil, as well as from the meat of the fish or from the whole fish.

“Another derivative of n-3 PUFA” as a compound for the second composition includes esters, amides, salts and phospholipids thereof, unless expressly stated otherwise. Examples for esters are alkyl esters, like C ₁-C ₆-alkyl esters, preferably C1-C4 alkyl esters, more preferably methyl or ethyl esters. Particularly, the alkyl esters are ethyl esters. Particularly preferred are ethyl esters of EPA and/or DHA. Examples for amides are alkyl amides, like C ₁-C ₆ mono or di-alkyl amides, preferably the amides are mono-N- (C1-C4 alkyl) amides or di-N, N- (C1-C4 alkyl) amides. Examples for salts are inorganic salts such as alkali salts, preferably sodium or potassium salts, earth alkali salts such as calcium or magnesium. Examples for salts further includes organic salts such as ammonium salts, salts formed with tartaric acid, acetic acid, propionic acid, citric acid, oxalic acid, malonic acid, salicylic acid, malic acid, fumaric acid, succinic acid, ascorbic acid, maleic acid, gluconic acid or lactic acid. Further examples are known to the person skilled in the art. The term further includes phospholipids. Preferably, phospholipids are phosphoglycerides or sphingomyelins.

In a preferred embodiment said combination of a first and second composition does not comprise docosahexaenoic acid (DHA) and polydatin.

In one preferred embodiment, the n-3 PUFA is present in the second composition (a) in its free acid form, (b) as monoglyceride, diglyceride and/or triglyceride and/or (c) as a C1-C4 alkyl ester. It is particularly preferred that the n-3 PUFA is present in the second composition as monoglyceride, diglyceride and/or triglyceride.

Alternatively, it is particularly preferred that the n-3 PUFA is present in the second composition as a C1-C4 alkyl ester, most preferably as an ethyl ester.

The composition of fatty acids in mixtures is preferably determined on the basis of the triglycerides in accordance with Ph. Eur. 1352/2.4.29.

In one embodiment, the second composition comprises at least 40 %by weight of the n-3 PUFA (expressed as triglycerides) , and more preferably at least 45%, 55%, 65%, 75%, 85%, or 95%.

In one embodiment, the second composition comprises high concentrations of one fatty acid or a monoacylglyceride, diacylglyceride, triglyceride and/or another derivative of n-3 PUFA, preferably the fatty acid is EPA and/or DHA. For instance, the composition may comprise at least 80 %EPA and/or DHA such as at least 90 %, e.g., about 97 %EPA and/or DHA by weight %of the fatty acids therein.

In at least one embodiment, the second composition comprises at least 60 % (of at least one of EPA or a derivative thereof and DHA or a derivative thereof by weight of the fatty acids therein, such as at least 75 weight %, such as at least 80 %, such as about 84 %of at least one of EPA or a derivative thereof and DHA or a derivative thereof by weight of the fatty acids in the composition. In one embodiment, the composition comprises essentially no DHA. In another embodiment, the composition comprises essentially no EPA.

“The derivative of EPA and/or DHA” includes monoacylglycerides, diacylglycerides, triacylglycerides and/or another derivative of n-3 PUFA as defined above.

In some embodiments of the present invention, the weight ratio EPA: DHA of the composition ranges from about 1: 10 to about 10: 1, from about 1: 8 to about 8: 1, from about 1: 6 to about 6: 1, from about 1: 5 to about 5: 1, from about 1: 4 to about 4: 1, from about 1: 3 to about 3: 1, or from about 1: 2 to about 2: 1. In at least one embodiment, the weight ratio of EPA: DHA of the composition ranges from about 1: 2 to about 2: 1. In at least one embodiment, the weight ratio of EPA: DHA of the composition ranges from about 1: 1 to about 2: 1. In at least one embodiment, the weight ratio of EPA: DHA of the composition ranges from about 1: 2 to about 1: 8, from about 1: 2 to 1: 4, from about 1: 2 to about 1: 3.

In another embodiment, the composition is selected from specific mixtures of EPA and DHA, or derivatives thereof, such as selected from compositions comprising about 360 mg EPA and 240 mg DHA per g polyunsaturated oil, 400 mg EPA and 200 mg DHA per g oil, 500 mg EPA and 200 mg DHA per g oil, 150 mg EPA and 500 mg DHA per g oil, 460 mg EPA and 380 mg DHA per g oil, above 900 mg EPA per g oil, above 900 mg DHA per g oil, and 97%EPA.

In an alternative preferred embodiment, the combination for use according to the invention includes a second composition comprising an n-3 PUFA mixture of about 84 %by weight EPA and DHA, preferably comprising 460 mg EPA-ethyl ester and 380 mg DHA-ethyl ester per gram.

In one embodiment, the second composition comprises DPA, such as up to 5 %by weight, such as about 2 %by weight. Some omega-6 fatty acids may be present, such as arachidonic acid or γ-linolenic acid, but the content is preferably kept low.

The term “triglycerides” herein includes a triglyceride derived from esterification of glycerol with a single or a mixture of different fatty acids.

In a preferred embodiment, the second composition comprises:

a) more than 60 %by weight triacylglycerides;

b) less than 38 %by weight diacylglycerides;

c) 0.5 %to 3 %by weight monoacylglycerides;

d) less than 5 %by weight residual ethyl ester; and

e) less than 0.5 %by weight free fatty acids.

In a particularly preferred embodiment, the second composition comprises:

a) about 60 %to about 75 %by weight triacylglycerides;

b) about 20 %to about 38 %by weight diacylglycerides;

c) about 0.5 %to about 1.5 %by weight monoacylglycerides;

d) about 1 to about 4 %by weight residual ethyl ester; and

e) less than 0.2 %by weight free fatty acids.

Particularly preferred, the second composition comprises

150: 500 TG EU (BASF) . This is a fish oil concentrate comprising triglyceride (TG) rich in omega-3 fatty acids. Additionally, a tocopherol-rich extract mainly derived from soybean is present. In 1g oil 150 mg EPA (expressed as triglycerides) and 500 mg DHA (expressed as triglycerides) are present.

“A human patient suffering from non-alcoholic fatty liver disease (NAFLD) ” has the following meaning. NAFLD encompasses the entire spectrum of fatty liver disease (FLD) in individuals without significant alcohol consumption, ranging from fatty liver to hepatic steatohepatitis to cirrhosis. Non-alcohololic fatty liver (NAFL) is characterized by the presence of at least 5%hepatic steatosis without evidence of hepatocellular injury in the form of ballooning of the hepatocytes or evidence of fibrosis. Non-alcoholic steatohepatitis (NASH) is characterized by the presence of ≥5%hepatic steatosis with inflammation and hepatocyte injury (ballooning) with or without fibrosis. NASH cirrhosis is the presence of cirrhosis with current or previous histological evidence of steatosis or steatohepatitis.

In a preferred embodiment the disease to be treated is NAFL. In another preferred embodiment the disease to be treated is NASH.

In addition to imaging techniques, fatty liver can also be detected using computer tomography (CT) . Fat has a lower attenuation than water using X-ray based techniques. This makes the liver appear darker on images and, by measuring the radiodensity, fat can be quantified. The following techniques may be used for diagnosis: controlled attenuation parameter (CAP) , fatty liver index (FLI) , magnetic resonance imaging (MRI) , magnetic resonance spectroscopy (MRS) , NAFLD liver fat score, SteatoTest and ultrasound.

“Treatment” may be observed on the basis of an improvement of any of the following parameters. The improvement may reside in the liver enzymes ALT, AST, GGT; total cholesterol; LDL cholesterol; HDL-cholesterol and liver fat. Preferably, the liver fat content is reduced. This may be assessed by the use of magnetic resonance imaging (MRI) , controlled attenuation parameter (CAP) and ultrasound. Alternatively, it may be assessed using computer tomography (CT) .

The total daily dosage of the first composition may range from about 0.600 g to about 6.000 g phytosterol, phytostanol, phystosterol ester, phytostanol ester and/or another phytosterol derivative (based on the free phytosterol and/or free phytostanol) . For example, in some embodiments, the total dosage ranges from about 0.500 g to about 4.000 g, from about 1.000 g to about 4.000 g, such as 3.000 g, or from about 1.000 g to about 2.000 g.

The first composition may be in the form of a powder or in the form of an oil.

The total daily dosage of the second composition may range from about 0.600 g to about 6.000 g n-3 PUFA, a monoacylglyceride of n-3 PUFA, a diacylglyceride of n-3 PUFA, a triacylglyceride of n-3 PUFA and/or another derivative of n-3 PUFA (based on the free n-3 PUFA) . For example, in some embodiments, the total dosage thereof ranges from about 0.500 g to about 4.000 g, from about 1.000 g to about 4.000 g, such as 3.000 g, or from about 1.000 g to about 2.000 g.

The second composition may be in the form of an oil.

The combination of the first and second composition may be administered in from 1 to 10 dosages, such as from 1 to 4 times a day, such as once, twice, three times, or four times per day, and further for example, once, twice or three times per day. The administration of the first composition may be oral or any other form of administration that provides a dosage of a plant sterol, plant stanol, plant sterol ester and/or plant stanol ester to a subject.

In a preferred embodiment, the subject is administered with about 1 to about 2 g phytosterol, phytostanol, phytosterol ester, phytostanol ester (based on the free phytosterol or free phytostanol) per day. In one embodiment, the dose is adjusted according to the level of phytosterol/phytostanol, phytosterol ester and/or phytostanol ester measured for the individual patient. The first composition is preferably administered over a long period, such as 12-52 weeks, e.g. 24-46 weeks. An adequate level of phytosterol, phytostanol, phytosterol ester and/or phytostanol ester is expected to be reached after 12-16 weeks, but the patient should continue the treatment to maintain this level. In one embodiment, the patient should continue to take the composition for the rest of the life.

The administration of the second composition may be oral or any other form of administration that provides a dosage of n-3 PUFA, a monoacylglyceride, a diacylglyceride, a triacylglyceride and/or another derivative of n-3 PUFA to a subject.

In a preferred embodiment, the subject is administered with about 1 to about 2 g EPA, monoglyceride, diglyceride, triglyceride and/or another derivative thereof (based on the free form) and/or DHA, monoglyceride, diglyceride, triglyceride and/or another derivative thereof (based on the free form) .

In an alternative embodiment, the capsules comprises 460 mg EPA-ethyl ester and 380 mg DHA-ethyl ester. In one embodiment, the dose is adjusted according to the level of n-3 PUFAs measured for the individual patient. The composition is preferably administered over a long period, such as 12-52 weeks, e.g. 24-46 weeks. An adequate level of n-3 PUFAs is expected to be reached after 12-16 weeks, but the patient should continue the treatment to maintain this level. In one embodiment, the patient should continue to take the composition for the rest of the life.

In some embodiments of the present disclosure, the combination acts as an active pharmaceutical ingredient (API) . In some embodiments, the first and/or second composition comprises a pharmaceutically-acceptable amount. As used herein, the term "effective amount" means an amount sufficient to treat, e.g., reduce and/or alleviate the effects, symptoms, etc., at least one health problem in a subject in need thereof.

The composition may contain further active agents. Preferably, the additional active agents includes metformin, thiazolidinediones or glucagon-like peptide-1 analogs.

The pharmaceutical composition may further contain pharmaceutically acceptable carriers which are well-known in the art. Examples may be stabilizers, bulking agents and buffer components. Additionally, the composition may contain at least one fat-soluble vitamin selected from Vitamin A, D, E and K. Preferably, the vitamin is Vitamin D and/or Vitamin E.

In this embodiment, the combination may be used in a pharmaceutical treatment of patients diagnosed with NAFLD. When the combination is a pharmaceutical composition, the second composition preferably comprises at least 75%of at least one of EPA and DHA, monoacylglycerides, diacylglycerides, triacylglycerides and/or another derivative thereof by weight of the composition. For example, in one embodiment, the composition comprises at least 80%by weight of the composition, such as at least 85%, at least 90 %, or at least 95%by weight of the composition.

In this embodiment, the first composition will be separate from the above composition and comprise at least 75 %of at least one phytosterol, phytostanol, phytosterol ester and/or phytostanol ester, preferably at least 80 %, more preferably at least 85 %, or even more preferably at least 95 %of the composition.

In another embodiment, the first composition as defined above and/or the second composition as defined above of the combination according to the invention is a food supplement or a nutritional supplement.

In a related embodiment, the invention provides a composition selected from the group of Enteral Formulas for Special Medical Use, Foods for Specified Health Uses, Food for Special Medical Purposes (FSMP) , Food for Special Dietary Use (FSDU) , Medical Nutrition, and a Medical Food.

The composition is suited for a nutritional management of NAFLD patients having a distinctive nutritional requirement. Such a composition typically is administered to the subject under medical supervision. Accordingly, the composition for use in the treatment of a patient who has NAFLD is selected from the above group. In a preferred embodiment, the composition is, or forms part of, Medical Food suitable for administration to NAFLD patients. The composition and the method of the invention have the ability to correct a nutritional deficiency in a target population. The invention hence provides nutritional correction of risk factors associated with NAFLD. Also, when using a present composition in the nutritional management of NAFLD, e.g., as a Medical Food, it is preferred that the second composition of the combination is a highly concentrated composition of EPA and/or DHA, or derivatives thereof. Preferably, the second composition comprises at least 55%of at least one of EPA, monoglycerides, diglycerides, triglycerides and/or another derivative thereof and/or DHA, monoglycerides, diglycerides, triglycerides and/or another derivative thereof by weight of the composition. It is further preferred that the first composition of the combination is a highly concentrated composition comprising campesterol or its ester, stigmasterol or its ester and/or β-sitosterol or its ester. Preferably, the first composition comprises at least 55%of at least one of campesterol or its ester, stigmasterol or its ester and/or β-sitosterol or its ester (expressed as free phytosterol) by weight of the composition.

The food supplement may additionally include nutritionally acceptable carriers such as stabilizers, bulking agents and/or buffer components. The food supplement may include a fat-soluble vitamin selected from Vitamin A, D, E and/or K. Preferably, the vitamin is Vitamin D and/or Vitamin E.

The compositions presently disclosed may be administered, for example, in capsule, tablet, or any other drug delivery form. For example, the composition may be encapsulated, such as in a gelatin capsule. Formulated forms of each at the above are all encompassed by the definition of the composition. Examples of such formulations are Self Emulsifying Drug Delivery Systems which form an emulsion in an aqueous solution. For example, the composition may be in the form of a pre-concentrate of any of the above which spontaneously form an emulsion when mixed with gastric/intestinal fluid. Such emulsions, when formed, may provide for increased or improved solubility of the fatty acids for uptake in the body and/or provide increased surface area for absorption.

Further, the composition may be in the form of emulsions and formulations where the active/nutritional ingredient is microencapsulated or in the form of a gel or semi-solid formulations.

The invention is further described in the following examples which are solely for the purpose of illustrating specific embodiments of the invention, and are also not to be construed as limiting the scope of the invention in any way.

Examples

Combined effect of PUFA and Phytosterol Ester on NAFLD Subjects: A double-blind Placebo-controlled clinical trial

Table 1 provides an overview of the clinical trial

Inclusion criteria for the clinical trial

The current study utilized a double-blind, randomized-controlled trial. Adult participants (aged 32～65) diagnosed with mild to severe NAFLD using B-ultrasound in a previous physical examination, and with a liver-to-spleen attenuation ratio of ≤1.2 or liver attenuation ≤ 52 HU, as determined by unenhanced computerized tomography (CT) in the present study (see Boyce et al., AJR Am. J. Roentgenol. Vol. 194 No. 3 (2010) , 623-628) , and dyslipidemia (total cholesterol (TC) ≥ 5.2 mmol/L, low-density lipoprotein cholesterol (LDL-C) ≥ 3.36 mmol/L, or triglycerides (TG) ≥ 1.7mmol/L) or overweight (BMI ≥ 24 kg/m2) were recruited.

Exclusion criteria for the clinical trial

The following were exclusion criteria for the trial: pregnant; cardiovascular disease; cancer; disability; lactose intolerance; diabetes mellitus; excessive alcohol consumption (≥30 g/day for men and ≥20 g/day for women) ; hepatitis B or C or other liver diseases; taking hypoglycemic or lipid-regulating drugs (statins, fibrates) or other drugs that may impact glucose and lipid metabolism. The other exclusion factors were diseases that impact the participant’s metabolism or ability to participate in this study; for example, hyperthyroidism, mental disorder, or diseases associated with serious dysfunction of heart, liver, kidney. All subjects underwent comprehensive physical examinations and routine biochemical analyses of blood. The loss criteria were as follows: 1) intent to withdraw; 2) fail to attend follow-up; 3) elevated transaminase enzymes >3-fold above the upper normal limits; 4) reduced glomerular filtration rate ≥1 stage, as determined by the Kidney Disease Outcomes Quality Initiative criteria; 5) unable to tolerate adverse events from soy milk products; and 6) less than 80%compliance per-visit.

Study design

A total of 103 subjects of Chinese origin (Han Chinese) who were diagnosed with fatty liver by ultrasound at the Physical Examination Center of The First People's Hospital of Ningyang County, Tai'an City, Shandong Province were recruited for this study. The diagnosis of participants was verified with unenhanced CT before stratified randomization. Seven participants did not meet the inclusion criteria. Ninety-six subjects were enrolled, and 21 subjects withdrew from the study because of personal reasons, e.g., business trips. Seventy-five people participated in the whole trial (66 men and 9 women) (as shown in Table 1) .

Participants were randomly assigned to the following four groups: the PS group (N=16) received 3.3 g/day of phytosterol ester (PSE) -enriched soymilk (equal to 2g free phytosterol) and placebo capsules (vegetable oil blend: 30%palmitic acid, 40%oleic acid and 20%linoleic acid obtained by blending canola (rapeseed) , sunflower and palm oils) ; the FO group (N=21) received highly concentrated omega-3 polyunsaturated fatty acid triglycerides (EPA: DHA =150: 500, a total of 2g omega-3 content in 6 capsules/day) and regular soymilk powder; the PS+FO group (N=17) received 3.3 g/day of PSE-enriched soymilk plus a total of 2g omega-3 polyunsatured fatty acid triglycerides as defined above ; and the PO group (N=21) received the placebo treatment of regular soymilk and placebo capsules (as shown in Table 1) . All soymilk powders and capsules had similar shapes and sizes. The omega-3 PUFAs, PSE-enrichted soymilk and corresponding placebo samples were supplied by the company BASF. Bottles were given to the participants at the beginning and in the middle of the intervention period. Compliance was assessed based on recycled packages at every visit and the EPA+DHA concentration determination at the end of the trial. None of the participants were encouraged to specifically modify their lifestyles (including dietary habit) during the entire study period considering the compliance, but were instructed to refrain from consuming supplements or other products claiming to reduce blood cholesterol. Participants, care providers, those assessing outcomes are blinded.

Anthropometric and biochemical measurements

Anthropometrical, biochemical and CT scanning data were collected at baseline (the initiation of the trial) and after 12 weeks (the end of the trial) . BMI was calculated as body weight in kilograms divided by the square of height in meters. Waist circumference was measured at the midpoint between the inferior costal margin and the superior border of the iliac crest on the mid axillary line. Blood samples were drawn after an overnight fast and immediately centrifuged. Serum TC, TG, high-density lipoprotein cholesterol (HDL-C) , LDL-C, glucose levels, as well as the liver function and kidney function were determined using Hitachi 7600 analyzer (Hitachi, Ltd. Tokyo, Japan) .

CT examination

CT scanning was performed at the beginning and the end of the trial by the same doctor. The liver/spleen attenuation ratio was measured with non-enhanced scans (SOMATOM Definition AS) , and the presence of steatosis was indicated by a ratio of ≤1.2 or by a liver attenuation of ≤52HU (see Boyce et al., ibid) .

Cytokine determination

The serum levels of high sensitive-C reactive protein (hs-CRP; Cat. No: RAP002, BioVendor; 1: 1000 dilution) , transforming growth factor-beta (TGF-β; cat no: ELH-TGFb1, RayBiotech; 1: 20 dilution) , tumor necrosis factor-alpha (TNF-α) , interleukin-6 (IL-6) and interleukin-1 alpha (IL-1α; ProcartaplexTM Multiplex Immunoassay) in all patients from the three study groups and the control group were retrospectively measured at the beginning and end of the trial.

Serum DHA and EPA concentration analysis

1) Sample preparation

A total of 100 μl of serum, 10 μl of C19: 0 methyl ester internal standard solution (1 mg/ml) and 1 ml of 5%sulfuric acid-methanol were mixed by vortexing. The nitrogen was purged for 10s, and the vessel was sealed and reacted at 80℃ for 90 minutes. Water and n-hexane were sequentially added and mixed for 10 minutes to obtain the fatty acid methyl esters. The organic phases were evaporated to dryness under N2 gas, and the samples were taken up into n-hexane (100μl) prior to gas chromatography (GC) analysis.

2) GC analysis of the fatty acid profiles in serum

GC analysis was performed using a gas chromatograph (GC-2010, SHIMADZU, JAPAN) . Separation was performed on a capillary column DB-5ms (30m×0.25mm×0.25μm) . Nitrogen was used as the carrier gas at a flow rate of 1 mL/min. The temperature of the injector was 270℃, and the split ratio was 1: 1. The following temperature program was employed: (1) 70℃ for 5 minutes; (2) increase to 200℃ at 25℃/min; (3) increase to 240℃ at 2℃/min. A flame ionization detector (FID) was used, and the temperature was 320℃.

Statistical analysis

All parameters are expressed as the mean±standard deviation (SD) . Analysis was performed with the SPSS statistical package (SPSS, 17.0) . All parameter changes within each group during the study were evaluated by a paired t-test. Between-group comparisons of the rates of the changes in all parameters from baseline to 12 weeks were statistically analyzed by one-way ANOVA, followed by LSD multiple comparison tests. The patients with abnormal TG, TC and/or LDL-C were included in the subgroup and analyzed in the same way. The patients whose liver/spleen attenuation ratio≤1or with minimum value were included in the two subgroups and analyzed in the same way respectively. The criterion for significance was p<0.05.

Results

Ninety-six subjects were assigned to three study groups and a control group according to the average ratio of liver attenuation/spleen attenuation via stratified randomization. Each group included 24 subjects. However, 3 patients were loss from the control group and from the FO group, respectively, 8 patients were loss from the PS group, and 7 patients were loss from the PS+FO group because of poor compliance. During this trial, none of the patients developed cancer or cardiovascular events. The average age of patients in the PO, FO, PS and PS+FO groups ranged from 44 to 47 years. The groups were similar with respect to their baseline biochemical characteristics (Tables 2 and 3) .

Table 2 Comparison of the baseline characteristics, biochemical results and lipid profiles of the groups (n = 75)

^aP＜0.05, vs. FO groub; ^bP＜0.05, vs. PS group; P=0.125, bs. PO group

Table 3 The percentage of subjects with abnormal characteristics at baseline in each groups (n = 75)

As shown in Table 4, compared with the beginning of the trial, there was a minor decreasing in BMI at the end of the trial within each group.

Table 4 Changes in the characteristics and biochemical results between baseline and after 12 weeks of intervention (n = 75)

The extent of this reduction was similar among the four groups (data not shown) . This minor reduction in BMI may be due to the change in season: the baseline data were determined in winter in January 22, 2018 and the final data were determined in Spring in April 15, 2018. Regarding the blood lipid levels, compared with baseline, the TG levels of the FO and PS+FO groups showed decreasing trends at the end of the trial (Table 4) , while the TG levels in the PO and PS groups showed increasing trends over the course of the trial. The TG of the PS+FO group was reduced by 11.57%± 39.38, which was the largest reduction among the groups (Figure 1A) . The TC levels of the FO, PO and PS+FO groups showed decreasing trends over the course of the intervention (Table 4) . The TC of the PS+FO groups was reduced by 9.55%±16.13, which was the largest reduction among the groups (Figure 1 B) . In addition, compared with baseline, the LDL-C levels of the PS, PO and PS+FO groups showed decreasing trends at the end of the trial (Table 4) . However, the LDL-C level of the PO group decreased more than that of the other groups (Figure 1C) . In the present study, the PS intervention significantly increased the level of HDL-C compared with the placebo (P=0.009) , FO (P=0.05) and PS+FO (P=0.001) treatments within the HDL-C normal range (Figure 1 D) . Moreover, we analyzed the changes in the blood lipid profiles of the subgroups in which all the subjects exhibited hyperlipidemia (as shown in Figure 1 E-H) . This analysis showed that the lipid profiles were similar among these subjects because of the high incidence of hyperlipidemia in this trial.

Transaminases

The patients we recruited in the present study exhibited mild to moderate steatosis, and there were no significant differences in the levels of alanine aminotransferase (ALT) , gamma-glutamyl transferase (GGT) , and aspartate aminotransferase (AST) at baseline and after 12 weeks of intervention within/among groups and within/among subgroups with liver/spleen attenuation of less than 1 (Tables 2 and 4) . But we specifically keep an eye on the change of those patients that exhibit abnormal liver function at base line. Figure 2 (A-H) illustrates the change of ALT and GGT before and after 12 weeks of intervention for each of the patients that reported abnormal liver function in the present study. In fact, there were five, eight, seven, six patients exhibited abnormal ALT activity at base line in PS, FO, PS+FO and PO group, respectively. Correspondingly, two, six, five, three patients were reported different degree of decline at the end of intervention, which occupied 40%, 75%, 71%, 50%, respectively in each group (Figure 2A-D) . In addition, there were nine, nine, eight, ten patients exhibited abnormal GGT activity at base line in PS, FO, PS+FO and PO group, respectively. Correspondingly, four, five, eight, seven patients were reported different degree of decline at the end of intervention, which occupied 44%, 56%, 100%, 70%, respectively in each group (Figure 2E-H) . These data also showed that the combination of PS with FO seemed more effectively improve the liver function after 12 weeks supplementation.

Other biochemical markers

Serum levels of hs-CRP, TNF-α, TGF-β, IL-6 and IL-1α were retrospectively measured in all patients from the three study groups and the control group at the beginning and the end of the trial. The levels of hs-CRP, IL-6 and IL-1α did not significantly change throughout the trial within and among the groups. However, compared with the beginning of the trial, the TGF-β levels of the three study groups were significantly decreased at the end of the trial (PS group, P=0.000; FO group, P=0.002; PS+FO group, P=0.001) . The TNF-α levels were also significantly decreased in the FO group (P=0.036) , PS+FO group (P=0.005) and PO group (P=0.032) at the end of the intervention. Notably, TGF-β was significantly more reduced in the PS+FO group than in the PO group (P=0.032) (Figure 3 A) . TNF-αwas slightly more reduced in the PS+FO group than in the other three groups (Figure 3 B, P=0.072 vs. PS group, P=0.318 vs. FO group, P=0.511 vs. PO group) .

Hepatic steatosis measured by unenhanced CT

Considering the regional characteristics of hepatic steatosis, the person performing the CT scan selected three points on the right lobe of the liver, two points on the left lobe of the liver, and the caudate lobe of the liver. According to a previous study, patients with liver attenuation ≤ 52 HU and/or liver-to-spleen attenuation ratio ≤ 1.2 were included in the present experiment (Boyce et al., ibid) . As shown in Table 2, in this trial, the mean liver attenuation ranged from 45 to 48 HU, the ratio of average liver/spleen attenuation ranged from 1.01 to 1.08, and the ratio of minimum liver/spleen attenuation ranged from 0.88 to 0.97. After 12 weeks of intervention, patients in the PS+FO group showed improvement compared with baseline. At the end of the trial, the Δliver/spleen attenuation (the mean of the minimum value) , Δliver/spleen attenuation (the mean of all subjects) , Δliver/spleen attenuation (the mean of the subjects ≤1) and Δliver attenuation (HU) of the PS+FO group were 0.16±0.19 (P=0.090 vs. before intervention) , 0.14±0.25 (P=0.155 vs. before intervention) , 0.21±0.31 (P=0.083 vs. before intervention) and 1.75±9.93 (P=0.633 vs. before intervention) , respectively (Table 4) . The increasing rate of liver/spleen attenuation (the mean of all subjects) , liver/spleen attenuation (the mean of the minimum value) , liver/spleen attenuation (the mean of the subjects ≤ 1) and liver attenuation (HU) in PS+FO group were 19%± 38 (P= 0.069 vs. PS group, 4%±19; P=0.179, vs. FO group, 9%±21; P=0.220 vs. PO group, 10%±17) (Figure 4 A) , 24%±35 (P= 0.043 vs. PS group, 6%±22; P=0.158, vs. FO group, 11%±23; P=0.166 vs. PO group, 12%±18) (Figure 4 B) , 36%±55 (P= 0.144 vs. PS group, 8%±17; P=0.222, vs. FO group, 18%±27; P=0.231vs. PO group, 16%±25) (Figure 4 C) , and 9.6%±10.8 (P= 0.054vs. PS group, -4.6%±9.1; P=0.476, vs. FO group, 4.8%±9.2; P=0.325 vs. PO group, 2.9%±7.6) (Figure 4 D) , respectively. Notably, the increasing rate of liver/spleen attenuation in the subgroup of PS+FO that less than 1 was 36%±55, which seemed obviously higher than that in the other three groups. The above mentioned data implied that the combination of PS with omega fatty acid may effectively improve hepatic steatosis in NAFLD, especially for patients with moderate to severe NAFLD. The average liver/spleen attenuation was increased from 0.57±0.11 before PS+FO intervention to 1.11±0.14 after 12 weeks intervention.

Table 5: CT Change Rate Average

Treatment Group	Mean	%increase after intervention
FO	9	46.6
PS	4	20.7
PS+FO	19.3	100

Table 6: CT Change Rate Minimum

Treatment Group	Mean	%increase after intervention
FO	11	45.8
PS	6	25.0
PS+FO	24	100

Table 7: Liver/Spleen (ratio<1) increasing

Treatment Group	Mean	%increase after intervention
FO	18	50.0
PS	8	22.2
PS+FO	36	100

Tables 5 to 7 demonstrate a synergistic effect of the co-administration of the phytosterol ester in combination with the n-3 PUFA over the administration of the compounds alone.

Compliance and adverse effects

To further observe the metabolic status of the subjects after the consumption of FO, this study determined the serum levels of DHA and EPA before and after intervention. Before the intervention, there was no significant difference in the serum DHA and EPA concentrations of the groups (P>0.05; as shown in Table 2) . After 12 weeks of intervention, the concentrations of DHA and EPA in the serum of the FO group and PS+FO group were significantly higher than those before the intervention (P<0.001) (Table 4) . Compared with baseline data, the serum DHA and EPA concentrations in the FO group and PS+FO group increased by 1.14-and 2.22-fold, respectively, and the serum DHA and EPA concentrations in the PS+FO group increased by 1.65-and 2.21-fold, respectively (Table 2 and Table 4) . The concentrations of DHA and EPA in the FO group and PS+FO group were significantly higher than those in the groups that did not consume FO (PS group and PO group) after 12 weeks of intervention (P<0.001) (Figure 4A-D) . These results may partially verify good compliance in this trial.

No other adverse clinical or laboratory event associated with the use of PS and/or omega-3 fatty acids were observed or reported.

Claims

A combination comprising a first composition comprising a sterol, a stanol, a sterol ester, a stanol ester and/or another sterol derivative and together or separately a second composition comprising a n-3 polyunsaturated fatty acid (n-3 PUFA) , a monoacylglyceride of n-3 PUFA, a diacylglyceride of n-3 PUFA, a triacylglyceride and/or another derivative of n-3 PUFA for use in the prevention and/or treatment of a human patient suffering from non-alcoholic fatty liver disease (NAFLD) .
The combination for use according to claim 1, with the proviso that said combination does not comprise docosahexaenoic acid (DHA) and polydatin.
The combination for use according to claim 1 or 2, wherein NAFLD is selected from non-alcoholic fatty liver (NAFL) or non-alcoholic steatohepatitis (NASH) .
The combination for use according to any one of claims 1 to 3, wherein the human patient is characterized by hepatic steatosis.
The combination for use according to claim 4, wherein hepatic steatosis is characterized by a liver fat content of 5%or more, preferably the liver fat content is determined by ultrasound sonography, computer tomography (CT) scan, magnetic resonance imaging-proton density fat fraction imaging (MRI-PDFF) or fibroscan.
The combination for use according to any one of claims 1 to 5, wherein in the first composition the sterol is a phytosterol and/or the stanol is a phytostanol.
The combination for use according to claim 6, wherein the phytosterol or phytostanol comprises campesterol, stigmasterol, β-sitosterol, stigmastanol, campestanol, brassicasterol, lupeol and/or cycloartenol, preferably the phytosterol comprises campesterol, stigmasterol and/or β-sitosterol.
The combination for use according to any of claims 1 to 7, wherein the sterol ester or stanol ester is obtained by esterification of campesterol, stigmasterol, β-sitosterol, stigmastanol, campestanol, brassicasterol, lupeol and/or cycloartenol, preferably by esterification of campesterol, stigmasterol and/or β-sitosterol.
The combination for use according to any one of claims 1 to 8, wherein the sterol ester and stanol ester is derived from the esterification of the sterol or stanol with a single fatty acid or mixed fatty acids.
The combination for use according to claim 9, wherein the fatty acid or fatty acids is/are from natural origin or obtained from natural origin.
The combination for use according to any of claims 1 to 10, wherein in the second composition the n-3 PUFA comprises hexadecatrienoic acid (HTA) , α-linolenic acid (ALA) , stearidonic acid (SDA) , eicosatrienoic acid (ETE) , eicosatetraenoic acid (ETA) , eicosapentaenoic acid (EPA) , heneicosapentaenoic acid (HPA) , docosapentaenoic acid (DPA) , docosahexaenoic acid (DHA) , tetracosapentaenoic acid and/or tetracosahexaenoic acid (nisinic acid) .
The combination for use according to any of claims 1 to 11, wherein the n-3 PUFA is EPA and/or DHA.
The combination for use according to any of claim 1 to 12, wherein the monoglyceride, diglyceride and/or triglyceride of n-3 PUFA has at least one n-3 PUFA moiety.
The combination for use according to claim 13, wherein the triglyceride of the n-3 PUFA has at least one EPA moiety and/or at least one DHA moiety.
The combination for use according to any one of claims 1 to 14, wherein the another derivative of n-3 PUFA is selected from an ester, an amide, a salt and/or a phospholipid.
The combination for use according to claim 15, wherein the ester is a C1-C4 alkyl ester, preferably a methyl or ethyl ester, and more preferably an ethyl ester.
The combination for use according to claim 15, wherein the amide is an alkyl amide, preferably the alkyl amide is a mono-N- (C1-C4) alkyl amide or a di-N, N- (C1-C4) alkyl amide.
The combination for use according to claim 15, wherein the salt is an alkali or earth alkali salt, preferably the salt is a sodium or potassium salt.
The combination for use according to claim 15, wherein the phospholipid is a phosphoglyceride or phosphomyelin.
The combination for use according to any of claims 1 to 19, wherein the first composition and/or the second composition additionally comprises a vitamin.
The combination for use according to claim 20, wherein the vitamin is a fat-soluble vitamin selected from Vitamin A, D, E and K, preferably the vitamin is Vitamin D and/or Vitamin E.
The combination for use according to any of claims 1 to 21, wherein the treatment provides an increased reduction in liver fat compared to the administration of the second composition alone.
The combination for use according to claim 22, wherein the administration of the first composition and the second composition provides a synergistic reduction in liver fat.
The combination for use according to any of claims 1 to 23, wherein a total daily dosage of the first composition is from about 0.600 g to about 6.000 g phytosterol, phytostanol, phytosterol ester and/or phytostanol ester (based on the free phytosterol or free phytostanol) , and a total daily dosage of the second composition is from about 0.600 g to about 6.000 g n-3 PUFA, a monoacylglyceride of n-3 PUFA, a diacylglyceride of n-3 PUFA and/or a triacylglyceride of n-3 PUFA (based on the free n-3 PUFA) , preferably the total daily dosage is administered by once, twice or three times daily administration.
A method for the treatment of a human patient suffering from NAFLD comprising administering to said patient an effective amount of the first composition as defined in any one of claims 1 to 10 and an effective amount of the second composition as defined in any one of claims 11 to 19.
The method according to claim 25, with the proviso that the patient is not administered docosahexaenoic acid (DHA) in combination with polydatin.
The method according to claim 25 or 26, wherein NAFLD is selected from non-alcoholic fatty liver (NAFL) or non-alcoholic steatohepatitis (NASH) .
The method according to any one of claims25 to 27, wherein a total daily dosage of the first composition is from about 0.600 g to about 6.000 g phytosterol, phytostanol, phytosterol ester, phytostanol ester and/or another phystosterol derivative and/or phytostanol ester (based on the free phytosterol and/or free phytostanol) , and a total daily dosage of the second composition is from about 0.600 g to about 6.000 g n-3 PUFA, a monoacylglyceride of n-3 PUFA, a diacylglyceride of n-3 PUFA, a triacylglyceride of n-3 PUFA and/or another derivative of n-3 PUFA (based on the free n-3 PUFA) is administered.
The method according to claim 28, wherein a total daily dosage of the first composition is from about 1 g to about 3 g phytosterol ester (based on the free phytosterol) , and a total daily dosage of about 0.5 g to about 1.5 g of EPA, a monoglyceride, diglyceride, a triglyceride and/or another derivative thereof and/or DHA, a monoglyceride, diglyceride, a triglyceride and/or another derivative thereof (based on the free EPA and/or DHA) is administered.
The method according to claim 29, wherein in addition 300 IU to 1000 IU Vitamin D and/or 200 to 1500 IU Vitamin E are administered.
The method according to any one of claims 26 to 30, wherein the treatment provides a reduction in liver fat compared to the administration of the second composition alone.
The method according to claim 31, wherein the administration of the first composition and the second composition provides a synergistic reduction in liver fat, preferably the treatment effect is measured by CT or MRI.
A kit for pharmaceutical use comprising a first composition composition comprising campesterol or an ester thereof, stigmasterol or an ester thereof and/or β-sitosterol or an ester thereof, and a second composition comprising EPA or a monoacylglyceride, diacylglyceride, triacylglyceride or another derivativethereof and/or DHA or a monoacylglyceride, diacylglyceride, triacylglyceride or another derivative thereof.
The kit according to claim 33, with the proviso that said kit does not comprise docosahexaenoic acid (DHA) and polydatin.
The kit according to claim 33 or 34, wherein

(ii) the first composition comprises

a) a total content of 50 to 90 %sterol ester;

b) 10 %to 40 %campesterol (determined as free sterols in the sterols profile) ;

c) 1 %to 5 %campestanol (determined as free stanols in the sterols profile) ;

d) 10 %to 30 %stigmasterol (determined as free sterols in the sterols profile) ;

e) 10 %to 80 %β-sitosterol (determined as free sterols in the sterols profile) ;

f) 2 %to 15 %β-sitostanol (determined as free stanols in the sterols profile) ; and

ii) the second composition comprises

a) 50 %to 90 %total content of triglycerides; and

b) 20 %to 90 %triglycerides comprising at least one fatty acid moiety derived from EPA and/or DHA.
The kit according to any one of claims 33 to 35, wherein the first composition and/or the second composition additionally comprises a vitamin.
The kit according to claim 36, wherein the vitamin is Vitamin D and/or Vitamin E.
A food supplement for the dietary management of a human patient suffering from NAFLD comprising a first composition as defined in any one of claims 33 to 37 and a second composition as defined in claim 33 or 37.
The food supplement according to claim 38, with the proviso that said food supplement does not comprise docosahexaenoic acid (DHA) and polydatin.
The food supplement according to claim 38 or 39, wherein the first composition and/or the second composition additionally comprises a vitamin.
The food supplement according to claim 40, wherein the vitamin is Vitamin D and/or Vitamin E.
Use of the first composition as defined in any one of claims 1 to 10 and the second composition as defined in any one of claims 11 to 24 in a food supplement for the dietary management of NAFLD.
Use according to claim 42, with the proviso that said food supplement does not comprise docosahexaenoic acid (DHA) and polydatin.