3-hydroxybutyric acid compounds for use in reducing liver fat

Abstract

To provide a compound for use in reducing liver fat in a subject.SOLUTION: The present invention provides a compound for use in reducing liver fat in a subject, where the compound is selected from: (i) (R)-3-hydroxybutyrate; (ii) an ester of (R)-3-hydroxybutyrate; and (iii) an oligomer obtainable by polymerizing (R)-3-hydroxybutyrate moieties; or a pharmaceutically acceptable salt or solvate thereof. The compound is particularly useful in a subject suffering from a fatty liver. The compound can be used to treat non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH) or non-alcoholic fatty liver (NAFL).SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to compounds for use in reducing liver fat in a subject and treating conditions associated with fatty liver, such as steatosis and liver disease. The present invention also relates to prophylactic treatment of a subject to avoid or reduce the risk of contracting a disease associated with fatty liver. [Background technology]

[0002] Fatty liver or steatosis is a term that describes the accumulation of fat in the liver. A subject has fatty liver when fat makes up at least 5% of the liver by weight.

[0003] Fatty liver in subjects can result from many different etiologies. Liver fat is associated with aging, high body mass index (BMI), elevated blood pressure, and type 2 diabetes. Increased liver fat is becoming increasingly common, especially in developed countries and in environments with high calorie intake, for example due to excessive consumption of alcohol or fatty foods. Fatty deposition in the liver can cause hepatomegaly, and fibrosis can result in the formation of scar tissue, which leads to liver hardening, reduced function, and ultimately failure. Thus, increased liver fat represents a major concern for public health.

[0004] Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease resulting from the accumulation of excess fat in the liver. It is a progressive liver disease that begins with the accumulation of fat in the liver without excessive alcohol consumption and is strongly associated with metabolic syndrome (obesity, insulin resistance, and dyslipidemia). NAFLD encompasses a range of pathological conditions, ranging from simple steatosis to nonalcoholic steatohepatitis, cirrhosis, and liver cancer. The disease is widespread and is the most common cause of chronic liver disease in Western countries. Approximately 20 to 30% of adults in the general population of Western countries have nonalcoholic fatty liver disease, and its prevalence increases to 70 to 90% among people who are obese or have diabetes. NAFLD also affects the pediatric population. Due to its close association with obesity, the condition has become the most common liver disease in children in the United States.

[0005] It is known that NAFLD is associated with increased morbidity and mortality from cardiovascular disease.In fact, increasing evidence shows that cardiovascular disease is the main cause of death in patients with advanced NAFLD, and this increased risk of cardiovascular disease is independent of the risk posed by the traditional risk factors and components of obesity-related metabolic syndrome.It is possible that NAFLD may be involved in the pathogenesis of cardiovascular disease.

[0006] Fatty liver is usually suspected in patients with abnormal liver function test results or hepatomegaly. Fatty liver can be diagnosed using needle biopsy of the liver. However, this procedure is painful and carries the risk of complications, so non-invasive methods for the assessment of liver fibrosis have been developed in the past decade. Roughly, non-invasive techniques can be divided into those based on direct and indirect serum markers of fibrosis and those based on imaging or electrogram recording. Magnetic resonance techniques can provide an attractive non-invasive option for liver evaluation.

[0007] Increased liver fat may also be observed in subjects who are not overweight and do not show any outward signs of liver disease. Public health can be improved by reducing liver fat to normal levels in subjects with increased liver fat, whether or not they are healthy, whether or not they have a disease associated with increased liver fat, and whether or not they are lean or overweight.

[0008] Currently, there is no effective approved treatment for the treatment of NAFLD.Typically, subjects are advised to lose weight, exercise, reduce or avoid alcohol intake, and ensure a balanced and healthy diet.Treatments may be recommended for individual components of metabolic syndrome, such as insulin sensitizers for diabetes.Weight loss may improve liver function test results if the subject is overweight or obese, but generally requires the subject to maintain proper exercise and dietary control, which can prove difficult for many reasons.

[0009] Thus, there is a need for new and effective treatments for reducing liver fat in subjects, particularly in subjects suffering from fatty liver.

[0010] The term "ketone bodies" is generally understood to encompass three compounds: D-β-hydroxybutyrate, acetoacetate, and acetone. D-β-hydroxybutyrate is alternatively known as (R)-3-hydroxybutyrate, and the latter term will be used below. Ketone bodies are produced by the liver from fatty acids during periods of low food intake.

[0011] Ketone bodies and ketone body esters have been shown to reduce serum cholesterol and / or triglyceride levels. For example, US Pat. No. 5,999,333 discloses a ketogenic diet that doubles plasma β-hydroxybutyric acid concentrations after administration. Total serum cholesterol, as well as HDL and LDL levels, were significantly lower in rats fed this ketogenic diet.

[0012] Given the difficulties in treating fatty liver, one would not have expected that a compound that is effective in lowering fat levels in serum, as discussed above, would also have the same effect in the liver.

[0013] In Non-Patent Document 1, the effect of ketones on cholesterol homeostasis was studied. Rats fed R-3-hydroxybutyl R-3-hydroxybutyrate showed reduced levels of acetoacetyl-CoA and HMG-CoA, precursors of mevalonate, in the liver, compared to controls. Hepatic levels of lanosterol and malonyl-CoA, precursors of fatty acid synthesis, were also lower. However, this reference does not disclose or suggest any effect on the level of fat in the liver.

[0014] US Patent No. 5,399,663 discloses the use of beta-hydroxybutyric acid mineral salts in healthy subjects to improve various metabolic biomarkers, including lowering blood glucose levels. This document specifically investigates the amount of weight gain in rats over a four-week period following dietary supplementation with ketones. The effect of ketone supplementation was found to reduce overall weight gain compared to control animals. The data in US Patent No. 5,399,663 shows that the liver weight of rats treated with ketone supplementation increased in proportion to the overall weight gain. US Patent No. 5,399,663 does not suggest that ketone supplementation may have efficacy in selectively targeting accumulated liver fat, for example in patients with steatosis.

[0015] Ketone bodies and ketone body esters have also been shown to have various other uses, such as treating muscle damage or fatigue and protecting against radiation exposure. Some of these compounds have also been tested in the liver. For example, some studies have suggested that D-β-hydroxybutyric acid can be used to prevent liver damage induced by lipid peroxidation (Non-Patent Document 2). However, these compounds have not been shown to reduce liver fat levels.

[0016] Patent Document 3 relates to ketone bodies and ketone body esters for maintaining or improving muscle power output. Example 5 uses a specific ester, R-3-hydroxybutyric acid-R-1,3-butanediol monoester, which is shown to increase blood R-3-hydroxybutyric acid concentrations to levels not previously reported for other ketones. Reduction of liver fat is not disclosed.

[0017] Patent Document 4 relates to a method for forming an oligomeric ketogenic compound. In paragraph

[0029] , this document discloses that this compound can be used to treat various neurological and psychiatric disorders. Again, there is no disclosure of reduction of liver fat.

[0018] Now, it has surprisingly been found that liver fat can be reduced in a subject, regardless of whether the subject has a disease associated with increased liver fat.The compounds of the present invention may be utilized to treat healthy subjects as well as subjects suffering from diseases associated with increased liver fat.Administration of the compounds of the present invention may also provide a preventive effect in reducing the risk of subjects suffering from diseases associated with fatty liver.The compounds of the present invention provide an effective treatment for fatty liver disease, including non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH) and non-alcoholic fatty liver (NAFL), liver cirrhosis, and hepatocellular carcinoma.As detailed above, until now, it has been very difficult to control these conditions in the clinic. [Prior art documents] [Patent documents]

[0019] [Patent Document 1] WO2009 / 089144 [Patent Document 2] WO2014 / 153416 [Patent Document 3] US2015 / 164855 [Patent Document 4] US2007 / 208081 [Patent Document 5] WO2004 / 108740 [Patent Document 6] WO2014 / 140308 [Non-patent literature]

[0020] [Non-Patent Document 1] Kemper et al., Lipids (2015) 50:1185-1193 [Non-Patent Document 2] Mol Endocrinol, August 2015, 29(8):1134-1143 [Non-Patent Document 3] “Metabolic Regulation: A Human Perspective” by KN Frayn [Non-Patent Document 4] Pavlides M et al, J Hepatol 2016; 64: 308-315 [Non-Patent Document 5] Thomas EL et al, Gut 2005; 54: 122-127 [Non-Patent Document 6] Handbook of Pharmaceutical Excipients, 2nd Edition, (1994), Edited by A Wade and PJ Weller Summary of the Invention [Problem to be solved by the invention]

[0021] Thus, the present invention provides a significant advantage to public health. [Means for solving the problem]

[0022] Thus, in a first aspect, the present invention provides a compound for use in reducing liver fat in a subject, the compound comprising: (i) (R)-3-hydroxybutyric acid; (ii) Esters of (R)-3-hydroxybutyric acid; and (iii) Oligomers obtainable by polymerizing (R)-3-hydroxybutyric acid moieties; or a pharma- ceutically acceptable salt or solvate thereof; is selected from.

[0023] In a second aspect of the invention, there is also provided a pharmaceutical composition for use in reducing liver fat comprising a compound as described in the first aspect of the invention and one or more pharma- ceutically acceptable excipients.

[0024] In a third aspect of the invention there is provided a nutritional composition for use in reducing liver fat comprising a compound as described in the first aspect of the invention and optionally further comprising water and one or more of flavourings, proteins, carbohydrates, sugars, fats, fibre, vitamins and minerals.

[0025] In a fourth aspect of the invention there is provided the use of a compound as described in the first aspect of the invention, or a composition according to the second or third aspect of the invention, in the manufacture of a medicament for use in reducing liver fat in a subject.

[0026] In a fifth aspect of the invention, there is provided a method of reducing liver fat in a subject comprising administering to the subject a compound as described in the first aspect of the invention or a composition according to the second or third aspect of the invention. [Brief description of the drawings]

[0027] [Figure 1] FIG. 1 shows the same weight loss in seven lean subjects (left) and three obese subjects (right) after taking 393 mg / kg body weight of (R)-3-hydroxybutyric acid-(R)-1,3-butanediol monoester (ΔG®) three times a day for five days. [Figure 2a]FIG. 10 shows no reduction in liver fat in seven lean subjects compared with reduction in liver fat in three obese subjects after taking 393 mg / kg body weight of (R)-3-hydroxybutyric acid-(R)-1,3-butanediol monoester (ΔG®) three times per day for five days (top panel). [Figure 2b] FIG. 1 shows histological images of normal liver and fatty liver together with their respective diagrams. [Figure 3a] 1H magnetic resonance spectrum of normal liver (lower panel) and fitting of the spectrum for quantification of liver fat (upper three panels). [Figure 3b] FIG. 1H magnetic resonance spectrum of fatty liver (lower panel) and fitting of the spectrum for quantification of liver fat (upper three panels). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] The compounds of the present invention provide a source of (R)-3-hydroxybutyric acid in the subject's body. Thus, the compounds may be (R)-3-hydroxybutyric acid itself or a precursor to (R)-3-hydroxybutyric acid, such as an ester or oligomer thereof, that can be broken down in the body to form (R)-3-hydroxybutyric acid.

[0029] (R)-3-hydroxybutyric acid is a ketone body and is defined in Non-Patent Document 3.

[0030] Patent document 5 discloses that ketone bodies can be directly administered to a subject to achieve elevated ketone body levels. However, direct administration can be difficult and dangerous under certain circumstances, and therefore the use of esters has been proposed as a preferred alternative. The production of ketone esters is disclosed, for example, in Patent document 6, which describes a process for producing (R)-3-hydroxybutyl (R)-3-hydroxybutyric acid.

[0031] Esters of (R)-3-hydroxybutyric acid can be produced via transesterification of ethyl-(R)-3-hydroxybutyrate with alcohols. This reaction may be enzymatically catalyzed. For example, the ethyl esters of (R)-3-hydroxybutyric acid and (R)-1,3-butanediol can be reacted together under mild reduced pressure in the presence of immobilized lipase to remove the resulting ethanol by-product.

[0032] In a preferred embodiment of the invention, the ester of (R)-3 hydroxybutyric acid has the general formula I:

[0033] [ka] is a compound of the formula - R1 is a C1-C6 alkyl group, which alkyl group carries up to 5 -OR2 substituents; - R2 represents hydrogen or C1-C6 alkyl, or -OR2 represents an (R)-3-hydroxybutyric acid moiety, or - R1 is a moiety derived from an alcohol HOR1, which alcohol is a sugar.

[0034] Typically, zero, one, or two -OR2 groups represent an (R)-3-hydroxybutyric acid moiety. Preferably, zero or only one -OR2 group represents an (R)-3-hydroxybutyric acid moiety.

[0035] Preferred compounds of the invention are esters, particularly those outlined above in formula I. The R1 moiety is derived from the corresponding alcohol HO-R1, which may be, for example, a monoalcohol, a diol, a polyol, or a sugar.

[0036] Preferably, in formula I, R1 is a C1-C6 alkyl group substituted with 0, 1, 2, 3, 4, or 5 -OR2 substituents. Most preferably, R1 is a C1-C6 alkyl group substituted with 1, 2, or 3 -OR2 substituents, typically 1 or 2 -OR2 substituents.

[0037] Preferably, R2 is H.

[0038] Preferably, R1 has the formula -CH2-CH(OH)-CH2(OH) or -CH2-CH2-CH(OH)-CH3. In these cases, R1 is a moiety derived from the alcohol HO-R1, which corresponds to butanediol and glycerol, respectively. Butanediol may be racemic 1,3 butanediol. Most preferably, the alcohol HO-R1 corresponds to R-1,3 butanediol. In this case, the group R1 has the formula:

[0039] [ka] It is of the following.

[0040] Preferably, the compounds of the invention are monoesters, i.e., when the alcohol HO-R1 contains two or more pendant hydroxyls, only one of these reacts to form a hydroxybutyric acid moiety. Partial esters are compounds where the alcohol HO-R1 contains two or more pendant hydroxyls, but not all of these react to form a hydroxybutyric acid moiety.

[0041] Particularly preferred compounds of the present invention have the formula:

[0042] [ka] It is (R)-3-hydroxybutyric acid (R)-1,3-butanediol monoester, also known as (R)-3-hydroxybutyl-(R)-3-hydroxybutyric acid.

[0043] Further preferred compounds of the invention are (R)-3-hydroxybutyric acid-glycerol partial esters, ie (R)-3-hydroxybutyric acid-glycerol monoesters or diesters.

[0044] In a different embodiment of the invention, R1 is obtained from an alcohol HOR1, which is a sugar. The sugar may be selected from altrose, arabinose, dextrose, erythrose, fructose, galactose, glucose, gulose, idose, lactose, lyxose, mannose, ribose, ribulose, sucrose, talose, threose, and xylose.

[0045] When R1 is derived from an alcohol HOR1 which is a polyol, the polyol may be selected from glycerol, ribitol and xylitol.

[0046] In an alternative embodiment of the invention, the compound of the invention has formula II:

[0047] [ka] is a compound of the formula - R1 is as described above in the first aspect of the invention, and further - n is an integer from 2 to 100.

[0048] Preferably, n is from 2 to 50, for example from 2 to 20, 2 to 10, or 2 to 5. The oligomer may, for example, contain only 2, 3, 4, or 5 repeat units (n=2, 3, 4, or 5). The oligomer may be linear or cyclic in nature.

[0049] In a preferred embodiment of the present invention, R1 has the formula -CH2-CH(OH)-CH2(OH) or -CH2-CH2-CH(OH)-CH3, i.e., the alcohol used to form the ester is glycerol or 1,3 butanediol. The butanediol may be racemic 1,3 butanediol or (R)-1,3 butanediol. Preferably, it is (R)-1,3 butanediol.

[0050] When compounds of the present invention contain chiral centers in addition to those depicted in the formula above, the compounds may exist in racemic or pure enantiomeric forms.

[0051] The compounds of the present invention may be present as physiologically compatible salts. For example, the sodium, potassium, calcium, or magnesium salts thereof may be utilized.

[0052] The inventors have found that (R)-3-hydroxybutyric acid-(R)-1,3-butanediol monoester and (R)-3-hydroxybutyric acid-glycerol partial ester provide high circulating levels of (R)-3-hydroxybutyric acid in blood and reduce liver fat.Furthermore, these esters provide surprisingly high levels of intestinal uptake, thereby allowing high blood levels of (R)-3-hydroxybutyric acid to be achieved upon consumption of the beverage.

[0053] Thus, in a preferred embodiment, the present invention provides hydroxybutyrate esters or partial esters, such as (R)-3-hydroxybutyric acid butane-1,3-diol monoester and (R)-3-hydroxybutyric acid glycerol partial ester, for use in reducing liver fat in a subject.

[0054] Particularly advantageous is (R)-3-hydroxybutyl-(R)-3-hydroxybutyric acid, because it allows for a greater increase in blood (R)-3-hydroxybutyric acid to be achieved with oral ingestion of a smaller amount of the substance than racemic ketones. A subject taking the substance can more easily ingest sufficient ketones to provide a physiologically beneficial response without the risk of physical discomfort (e.g., from the ingestion of large amounts of liquid or bitter / otherwise aversive flavors). (R)-3-hydroxybutyl-(R)-3-hydroxybutyric acid also elevates (R)-3-hydroxybutyric acid concentrations for a longer period of time than ketone salts. Thus, fewer doses are required to maintain elevated (R)-3-hydroxybutyric acid levels. This also facilitates subject compliance with the dosing regimen.

[0055] Administration of a compound of the invention to a subject can reduce the level of liver fat in the subject, advantageously reducing the risk of the subject developing disease-related increased liver fat or becoming susceptible to diseases associated with increased liver fat.

[0056] Liver fat refers to lipids stored in liver cells (hepatocytes). In this context, the term "lipids" includes fatty acids and their derivatives, including tri-, di-, and monoglycerides, as well as phospholipids. The term liver fat in the present invention does not include cholesterol or precursors of cholesterol biosynthesis, such as lanosterol and mevalonic acid, and the mevalonic acid precursors acetoacetyl-CoA and HMG-CoA. The term liver fat also does not include fatty acid synthesis precursors, such as malonyl-CoA. Preferably, the term liver fat refers only to tri-, di-, or monoglycerides, and most typically only to triglycerides.

[0057] Compounds of the present invention are particularly useful for targeted reduction of fat in liver, particularly targeted reduction of accumulated fat in liver.Therefore, the reduction of liver fat in the present invention is the intended result of treatment, and is not merely the inevitable result of the reduction of body fat in general, for example, in the treatment of obesity.In this targeted treatment, the amount of fat in liver can be reduced at a greater rate than the reduction of the level of fat in the whole body.

[0058] The compounds of the present invention are particularly useful for the treatment of subjects suffering from fatty liver. The level of fat in the liver can be measured by proton magnetic resonance spectroscopy ( ) to determine the percentage of liver fat using the methods described in Non-Patent Document 4 and Non-Patent Document 5. 1 1 H MRS). Using this method, normal levels of fat may be around 2%, while increased or high liver fat may be greater than 5%, and in some cases perhaps as high as 20-30%. In the present invention, "fatty liver" refers to a liver having a fat content of greater than 5% by weight.

[0059] Thus, the present invention provides a compound as described above for use in a subject having a liver with a fat content of at least 5% by weight, to reduce the level of liver fat.

[0060] Subjects will have different levels of liver fat according to their physiological functions.For example, different subjects may be considered healthy, but due to different body types, they may have significantly different BMI and different levels of liver fat.The present invention provides advantages in reducing liver fat in each case.

[0061] Preferably, for subjects with 5-10% liver fat by weight, the compound is administered at a level and regimen that reduces the level of fat by at least 2 percentage points, i.e., gives a final level of liver fat in the range of 3-8% or less. If the subject has a liver fat level of 10-15%, the level of fat is reduced by at least 5 percentage points, i.e., gives a final level of liver fat in the range of 5-10% or less. If the subject has a liver fat level of more than 15%, the level of fat is reduced by at least 5 percentage points. All percentages are given relative to liver weight.

[0062] In a further aspect, the present invention provides a compound, preferably an ester, as described in the first aspect of the present invention, or a composition according to the present invention, in treating a subject to reduce liver fat, said treatment comprising administering to the subject a compound or composition according to the present invention for 5 days, wherein the level of fat in the liver is reduced by at least 1 percentage point, preferably by at least 3 percentage points, and desirably by at least 5 percentage points. For example, if the level of fat in the liver starts at 15% by weight, the level of fat in the liver after treatment is less than 14% by weight, preferably less than 12% by weight, and desirably less than 10% by weight.

[0063] Typically, the subject is overweight and / or suffers from fatty liver.

[0064] Suitably, the compounds of the invention, preferably (R)-3-hydroxybutyric acid-(R)-1,3-butanediol monoester, are ingested at a level of at least 100 mg / kg body weight of ketones per day. Desirably, the ketone bodies or ketone body esters are ingested at a level sufficient to provide a plasma ketone level of at least 0.1 mM, preferably at least 0.2 mM, more preferably at least 1 mM, and optimally at least 2 mM. Suitably, the ketone bodies or ketone body esters are ingested at a level such that the plasma ketone level does not exceed 20 mM, preferably does not exceed 10 mM or 8 mM, and may not exceed 5 mM.

[0065] It has been found that the plasma level of ketones depends on the body weight of the individual, and that oral administration of at least 300 mg / kg body weight of (R)-3-hydroxybutyric acid-(R)-1,3-butanediol monoester provides a plasma concentration of about 1.5 mM (R)-3-hydroxybutyric acid, and administration of 500 mg / kg provides at least 3 mM (R)-3-hydroxybutyric acid. At a dose of 1 g per kg of subject body weight, the blood concentration of (R)-3-hydroxybutyric acid is preferably at least 4 mM, preferably 5 mM. Oral administration of 1.5 g of the monoester per kg of subject body weight provides a blood concentration of (R)-3-hydroxybutyric acid of preferably at least 7 mM, preferably at least 8 mM, particularly at least 9 mM. The dosing regimen includes that multiple beverages are consumed separately.

[0066] Blood levels of (R)-3-hydroxybutyrate may be determined by commercially available test kits, for example, (R)-3-hydroxybutyrate can be measured in whole blood using a handheld monitor and reagent strips (Precision Xtra, Abbott Diabetes Care, UK).

[0067] The compounds of the present invention are suitable for treating subjects with fatty liver, whether lean, overweight, obese or severely obese, i.e. subjects with a BMI below 25, between 25 and 29.9, between 30 and 39, and above 40, respectively, to reduce liver fat. The compounds are suitable for treating subjects with diabetes or pre-diabetes to reduce liver fat.

[0068] The compounds of the invention may be used to treat healthy subjects to reduce levels of liver fat, or to treat subjects with liver disease, such as fatty liver disease, including treating non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), and / or non-alcoholic fatty liver (NAFL).

[0069] Accordingly, one aspect of the invention provides a compound for use in the treatment or prevention of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), and non-alcoholic fatty liver (NAFL), wherein the compound is: (i) (R)-3-hydroxybutyric acid; (ii) Esters of (R)-3-hydroxybutyric acid; and (iii) Oligomers obtainable by polymerizing (R)-3-hydroxybutyric acid moieties; or a pharma- ceutically acceptable salt or solvate thereof; is selected from.

[0070] Typically, subjects with fatty liver or fatty liver disease are overweight, obese, or severely obese, for example subjects have a BMI of 25 to 29.9 (overweight), a BMI of 30 to 39.9 (obese), and a BMI of 40 or greater (severely obese). The present invention is particularly applicable to male subjects with a waist circumference of 94 cm (37 inches) and female subjects with a waist circumference of 80 cm (approximately 31.5 inches) or greater.

[0071] The compound of the present invention may be included in a nutritional composition. Preferably, the nutritional composition comprises water and a source of (R)-3-hydroxybutyric acid. Preferably, the composition comprises an ester of (R)-3-hydroxybutyric acid, a flavoring agent, and optionally one or more of protein, carbohydrate, sugar, fat, fiber, vitamins, and minerals. Preferably, the flavoring agent may comprise a fruit-based flavoring agent. In one embodiment, the flavoring agent is preferably bitter, such as coffee, chocolate, and cranberry. The bitter flavoring agent may be combined with other flavoring agents, such as fruit-based flavoring agents, such as grapefruit, raspberry, and cranberry.

[0072] The compositions useful in the present invention may contain mixtures of isomers of the compounds described above.

[0073] The composition is preferably organoleptically acceptable, meaning that the composition must have acceptable sensory properties of taste, color, feel, and odor.

[0074] The composition may include medium chain triglycerides (MCT). When present, the medium chain triglycerides are preferably of the formula CHR a -CH2R b -CH2R c (In the formula, R a , R b , and R c Preferably, tri-C6:0 MCTs are reported to be very rapidly absorbed by the gastrointestinal tract, and therefore, R a , R b , and R c is a fatty acid with a six-carbon backbone (tri-C6:0).

[0075] The composition of the present invention may contain L-carnitine or a derivative of L-carnitine. Examples of derivatives of L-carnitine include decanoylcarnitine, hexanoylcarnitine, caproylcarnitine, lauroylcarnitine, octanoylcarnitine, stearoylcarnitine, myristoylcarnitine, acetyl-L-carnitine, O-acetyl-L-carnitine, and palmitoyl-L-carnitine. When carnitine is used, the composition of the present invention preferably contains i) a ketone body, preferably a ketone monoester, more preferably (R)-3-hydroxybutyric acid monoester, and ii) L-carnitine or a derivative of L-carnitine, and optionally MCT.

[0076] When MCT and L-carnitine or its derivatives are utilized, preferably, MCT is emulsified with carnitine.Preferably, 10 to 500g of emulsified MCT is combined with 10 to 2000mg of carnitine, for example, 50g of MCT (95% tri-C8:0) is emulsified with 50g of mono- and diglycerides combined with 500mg of L-carnitine.Preferably, the level of the source of (R)-3-hydroxybutyric acid is greater than the level of MCT.

[0077] The composition according to the invention may be provided in any suitable form including a solid such as a powder, tablet, bar, confectionery product or granule, a liquid such as a beverage, gel, capsule or any other conventional product form. The composition may be a food product, a food supplement, a dietary supplement, a functional food or a nutraceutical, or an ingredient thereof.

[0078] Examples of food products into which the composition can be incorporated as an additive include snack bars, cereals, confectioneries, and probiotic preparations, including yogurt. Examples of beverages include soft drinks, alcoholic beverages, energy drinks, dry drink mixes, nutritional drinks, and herbal teas for infusion or herbal blends for brewing in water.

[0079] A functional food is a food ingredient, dietary supplement, or food product that is believed to provide medical or health benefits, including disease prevention and treatment. In general, functional food is specifically adapted to provide health benefits to consumers. Functional food typically contains micronutrients, such as vitamins, minerals, herbs, or phytochemicals, at levels higher than those found in corresponding regular food products. The levels are typically selected to optimize the intended health benefits of the functional food when taken as a one-off or as part of a course of diet plan or nutritional therapy.

[0080] The compounds of the present invention are typically formulated as nutritional supplements.

[0081] When in solid form, the composition suitably comprises at least 5% by weight of a compound of the invention, preferably an ester, more preferably at least 10% to 95% by weight of the composition. For example, where the composition is a dry powder intended for use with a liquid to provide a liquid composition, a level of dry composition of 15 to 30% by weight may be suitable, although a solid bar or product form will suitably comprise 30 to 95% by weight, especially 50 to 95% by weight of the composition.

[0082] When the composition is in solid form, it comprises the following components: - a diluent such as, for example, lactose, dextrose, saccharose, cellulose, corn starch, or potato starch; lubricants such as, for example, silica, talc, stearic acid, magnesium or calcium stearate, and / or polyethylene glycol; - binders such as, for example, starch, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinylpyrrolidone; - disintegrants such as starch, alginic acid, alginates or sodium starch glycolate; - Foaming agent; - dye; - fragrance; wetting agents, such as, for example, lecithin, polysorbates, lauryl sulfate; and / or - Career; The composition may further include one or more of:

[0083] If the composition is in liquid form, the composition suitably contains a compound of the invention at a level of at least 1% by weight, such as from 3 to 40% by weight of the liquid composition, but may be higher, such as up to 50% by weight of the composition, depending on whether the composition is intended to be taken as a single dose or in multiple smaller doses to achieve the desired blood ketone levels.

[0084] A composition in liquid form may comprise several liquid components that are suitably blended together, or may comprise liquid and solid components that are mixed with or dissolved in the liquid component as needed. In one embodiment, the dry composition comprising a ketone is diluted with a suitable liquid, such as, for example, water, fruit juice, yogurt, or milk, preferably in a dry composition to liquid ratio of 1:1 to 1:10, more preferably 1:3 to 1:7.

[0085] The compositions may be provided as liquid products in a ready-to-consume form, as desired, or as concentrates or pastes suitable for dilution at the time of use. Diluents for use with liquid compositions are preferably milk, fruit juice, or water.

[0086] If desired, the composition can be provided in an encapsulated form, so long as the encapsulation material and the amount used are suitable for safe human consumption.

[0087] In a further aspect, the present invention provides a kit comprising a compound, preferably an ester, according to the first aspect of the invention, or a composition according to the invention, and a ketone monitor and optionally instructions regarding the level of product to consume per unit body weight and a dosing regimen for reducing liver fat. Suitably, a user consumes the product and can then periodically test their plasma ketone levels to determine whether further ketone intake is required to reach or maintain a desired plasma ketone level.

[0088] One aspect of the present invention provides a compound of the present invention as described above in a pharmaceutical composition, together with one or more pharma- ceutically acceptable excipients.

[0089] The compounds of the present invention may be present as pharmaceutically acceptable salts. As used herein, a pharmaceutically acceptable salt is a salt with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acids include inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid, or nitric acid, as well as organic acids such as citric acid, fumaric acid, maleic acid, malic acid, ascorbic acid, succinic acid, tartaric acid, benzoic acid, acetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or p-toluenesulfonic acid. Pharmaceutically acceptable bases include alkali metal (e.g., sodium or potassium) and alkaline earth metal (e.g., calcium or magnesium) hydroxides, and organic bases such as alkylamines, aralkylamines, and heterocyclic amines.

[0090] The compound of the present invention may exist as a solvate. The term "solvate" refers to a complex or aggregate formed by one or more molecules of a solute, i.e., the compound of the present invention or its pharma- ceutically acceptable salt, and one or more molecules of a solvent. Such a solvate is typically a crystalline solid having a substantially fixed molar ratio of solute and solvent. Representative solvents include, for example, water, methanol, ethanol, isopropanol, and acetic acid. When the solvent is water, the solvate formed is a hydrate.

[0091] The compounds of the present invention have chiral centers.Therefore, the compounds of the present invention can be used in the form of racemates, enantiomers, or mixtures enriched in one or more stereoisomers.The scope of the present invention as described and claimed includes the racemic forms of the compounds of the present invention, as well as mixtures enriched in individual enantiomers and stereoisomers.

[0092] It will be appreciated that the term "or a pharma- ceutically acceptable salt or solvate thereof" is intended to include all permutations of salts and solvates, such as solvates of a pharma- ceutically acceptable salt of the compound of the invention.

[0093] The pharmaceutical composition of the present invention comprises the compound of the present invention mixed with one or more pharma- ceutically acceptable diluents, excipients, or carriers. Although the compounds of the present invention (including their pharma- ceutically acceptable salts, esters, and pharma-ceutically acceptable solvates) can be administered alone, they are generally administered in admixture with a pharmaceutical carrier, excipient, or diluent, especially for human treatment. The pharmaceutical composition may be for human or animal use in human and veterinary medicine.

[0094] Examples of such suitable excipients for the various different forms of pharmaceutical compositions described herein can be found in Non-Patent Document 6.

[0095] The compositions of the invention (both pharmaceutical and nutritional compositions) may include a pharma- ceutically acceptable adsorbent. Suitably, the adsorbent adsorbs the compounds of the invention within or on the adsorbent. Advantageously, the flavor (which may be palatably aversive) of the compounds is experienced by the user to a lesser extent than would be experienced if the same composition were consumed without the adsorbent. Preferably, the adsorbent comprises a lattice or voids capable of retaining the compounds of the invention. Any adsorbent used or known for use in food products may be utilized. Examples of suitable adsorbents include polymeric hydrogels, such as cross-linked polycarboxylate homopolymer or copolymer polymers, clathrates, cyclic oligosaccharides, such as cyclodextrins, and milk powder. The adsorbent may be present at any desired level according to the particular formulation, for example 5% to 80% by weight of the composition, such as 10 to 50% by weight.

[0096] Typically, the subject of the present invention is a mammal, such as a human.

[0097] Typically, the uses of the present invention will involve administering the compounds orally, parenterally, or intravenously, with oral administration being preferred.

[0098] The present invention also provides compounds, as described herein, in substantially pure form or together with one or more pharma- ceutically acceptable diluents or carriers, for use in methods of reducing liver fat in a subject.

[0099] As used herein, the term "substantially pure form" typically refers to a compound that is 50% or more pure, preferably 75% or more pure, more preferably 90% or more pure, even more preferably 95% or more pure, and most preferably 99% or more pure.

[0100] The following examples illustrate the invention.

[0101] Working Example The invention will now be described with reference to the following non-limiting examples.

[0102] Example - Liver 1 H Magnetic Resonance Spectroscopy (MRS) Three obese subjects (BMI 33 ± 3 kg / m2) were matched for age, sex, diabetes duration, treatment, and HBA1c. 2 ) and seven lean subjects (BMI 23±2 kg / m 2 ) for the quantification of fatty liver 1 1H-MRS scan was performed.

[0103] All subjects were weighed and placed on a 5-day treatment regimen, with each subject consuming a beverage containing 0.393 g / kg body weight of (R)-3-hydroxybutyric acid-R-1,3-butanediol monoester three times a day, up to a final volume of 3.3 ml / kg body weight. Table 1 below shows the beverage formulation.

[0104] [Table 1] Subjects were not asked to change their usual diet, but were asked to keep a food diary. Subjects lost 1.3 kg over 5 days, regardless of their starting weight. The weight loss results are shown in Figure 1. The weight loss in 7 lean subjects (Figure 1a) and 3 obese subjects (Figure 1b) was the same at 1.3 kg after 393 mg / kg body weight of (R)-3-hydroxybutyric acid-(R)-1,3 butanediol monoester (ΔG®) 3 times a day for 5 days.

[0105] To determine changes in liver fat, each subject was given a liver biopsy at the beginning and end of the 5-day period. 1 H-MRS was performed. 1 H-MRS measurements were performed on a 3T Siemens Tim Trio using a 6-channel anterior + 24-channel posterior phased array coil and a whole-body coil for signal reception. 1To acquire H-MRS, 8-ml voxels (2 × 2 × 2) were placed within the liver, avoiding gross vascular, biliary structures, and adipose tissue depots. Spectra were acquired with and without water suppression, and liver triglyceride content was calculated as a percentage of water (triglycerides / water × 100). A calibration pulse sequence was performed to evaluate the optimal water suppression pulse scaling factor. Individual coil signals were combined in Matlab using a specially written module. Water suppression scans were constructively averaged, including frequency correction. Spectra were quantified using the Advanced Method of Accurate, Robust and Efficient Spectroscopic (AMARES) fitting algorithm. Metabolite peaks (including lipids / triglycerides) were analyzed, and prior knowledge was used for all peak locations by using soft constraints. All peaks were fitted using a Lorentzian line fit.

[0106] result The present invention allows healthy and obese subjects to lose weight as demonstrated by the weight loss results (Figure 1). In lean subjects, no reduction in liver fat was observed (2.3% and 2.5%). In obese subjects, liver triglyceride levels were reduced by about 3.4% (from 17.5 to 14.1%) in 5 days. The results are plotted in Figure 2 along with images of normal / lean and fatty liver. It can be observed that there are islands of fat in the fatty liver images.

[0107] Figure 2a shows a 3.4% reduction in liver fat in three obese subjects and no reduction in liver fat in seven lean subjects after taking 393 mg / kg body weight of (R)-3-hydroxybutyric acid-(R)-1,3 butanediol monoester (ΔG®) three times a day for five days. Figure 2(b) illustrates the histology of normal and fatty livers with their respective diagrams. There is a clear difference in the histology of these samples.

[0108] In lean and obese subjects1 Representative examples of H-MRS spectra are shown in Figures 3a and 3b, respectively. Figure 3a shows the results of a 1H-MRS spectrum of a lean subject with a liver triglyceride level of 2.6%. 1 H-MR spectra of normal liver (lower panel). 1 H magnetic resonance spectra showing the fitting of the spectra for quantification of liver fat (top three panels).

[0109] FIG. 3b shows the results of a 16.1% liver triglyceride level in an obese subject. 1 H-MR MR spectrum of a fatty liver (lower panel). 1 H magnetic resonance spectra showing the fitting of the spectra for quantification of liver fat (top three panels).

[0110] As you can see, 1 H-MR can be used to diagnose subjects with fatty liver.

[0111] Item 1: A compound for use in reducing liver fat in a subject, comprising: (i) (R)-3-hydroxybutyric acid, (ii) Esters of (R)-3-hydroxybutyric acid, and (iii) Oligomers obtainable by polymerizing (R)-3-hydroxybutyric acid moieties; or a pharma- ceutically acceptable salt or solvate thereof; A compound selected from: Item 2: Esters of (R)-3 hydroxybutyric acid are represented by the general formula I [ka] is a compound of the formula - R1 is a C1-C6 alkyl group, the alkyl group having up to 5 -OR2 substituents, R2 representing hydrogen or a C1-C6 alkyl, or -OR2 representing an (R)-3-hydroxybutyric acid moiety, or - the compound according to item 1, wherein R1 is a moiety derived from an alcohol HOR1, the alcohol being a sugar. Item 3: The compound according to item 2, wherein R1 is a C1-C6 alkyl group substituted with 1, 2 or 3 -OR2 substituents. Item 4: The compound according to item 2 or 3, wherein R2 is H. Item 5: A compound according to any one of items 2 to 4, wherein R1 has the formula -CH2-CH(OH)-CH2(OH) or -CH2-CH2-CH(OH)-CH3. Item 6: The compound according to item 2, wherein R1 is a moiety derived from an alcohol HOR1, and the alcohol is a sugar selected from altrose, arabinose, dextrose, erythrose, fructose, galactose, glucose, gulose, idose, lactose, lyxose, mannose, ribose, ribulose, sucrose, talose, threose, and xylose. Item 7: The oligomer has the formula II [ka] is a compound of the formula R1 is according to any one of items 2 to 6, - the compound according to item 1, wherein n is an integer from 2 to 100. Item 8: formula [ka] 7. The compound according to any one of items 1 to 6, which is (R)-3-hydroxybutyric acid (R)-1,3-butanediol monoester. Item 9: The compound according to any one of items 1 to 8, for use in treating a subject suffering from fatty liver. Item 10: The target is 1 10. The compound according to item 9, having greater than 5% liver fat by weight as measured using H magnetic resonance spectroscopy (MRS). Item 11: The compound according to any of items 1 to 10, wherein the compound is administered to the subject at least once a day, and the level of fat in the liver is reduced by at least 1 percentage point in liver weight, preferably by at least 3 percentage points in liver weight, and desirably by at least 5 percentage points in liver weight, 5 days after the first treatment, compared to the level of fat in the liver before the first treatment. Item 12: A compound according to any of items 1 to 11 for use in treating liver disorders by reducing liver fat, preferably the disorder is fatty liver disease, most preferably the disorder is non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH) or non-alcoholic fatty liver (NAFL). Item 13: A compound according to any of items 1 to 12 for use in treating a subject who is overweight (having a BMI in the range of 25 to less than 30), who is obese (having a BMI in the range of 30 to less than 40), or who is severely obese (having a BMI of 40 or greater). Item 14: The compound according to any one of items 1 to 13, wherein the subject is diabetic or prediabetic. Item 15: A compound according to any of items 1 to 11 for use in reducing the level of fat in the liver of a healthy subject. Item 16: A pharmaceutical composition for use in reducing liver fat, comprising a compound according to any one of items 1 to 15 and one or more pharma- ceutically acceptable excipients. Item 17: A nutritional composition for use in reducing liver fat comprising a compound according to any one of items 1 to 15, and optionally further comprising water and one or more of flavourings, proteins, carbohydrates, sugars, fats, fibre, vitamins and minerals. Item 18: The method further comprises the step of: providing a medium chain triglyceride, and preferably the medium chain triglyceride is represented by the formula CHR a -CH2R b -CH2R c wherein R a , R b, and R c 18. The nutritional composition according to item 17, wherein is a fatty acid having 5 to 12 carbon atoms. Item 19: Use of a compound according to any one of items 1 to 8 or a composition according to any one of items 16 to 18 in the manufacture of a medicament for use in reducing liver fat in a subject, preferably according to any one of items 9 to 15. Item 20: A method for reducing liver fat in a subject, comprising administering a compound according to any one of items 1 to 8 or a composition according to any one of items 16 to 18 to a subject, preferably according to any one of items 9 to 15.

Claims

1. A pharmaceutical composition for oral administration for use in preventing or treating fatty liver disease, comprising as an active ingredient for reducing liver fat: (i) (R)-3-hydroxybutyric acid, (ii) esters of (R)-3-hydroxybutyric acid, and (iii) Oligomers obtainable by polymerizing (R)-3-hydroxybutyric acid moieties; or a pharma- ceutically acceptable salt or solvate thereof; A pharmaceutical composition for oral administration comprising a compound selected from the group consisting of:

2. The ester of (R)-3-hydroxybutyric acid is represented by the general formula I 【Chemistry 1】 is a compound of the formula - R １ is C １ ~C ６ wherein the alkyl group is an alkyl group having up to 5 -OR ２ has a substituent, R ２ is hydrogen or C １ ~C ６ or represents an alkyl group of the formula -OR ２ represents an (R)-3-hydroxybutyric acid moiety, or - R １ Alcohol HOR １ 2. The pharmaceutical composition for oral administration according to claim 1, wherein the alcohol is a sugar.

3. R １ is 1, 2 or 3 -OR ２ C substituted with a substituent １ ~C ６ The pharmaceutical composition for oral administration according to claim 2 , wherein the alkyl group is:

4. R ２ The pharmaceutical composition for oral administration according to claim 2 or 3, wherein is H.

5. R １ is of the formula -CH ２ -CH(OH)-CH ２ (OH) or -CH ２ -CH ２ -CH(OH)-CH ３ 5. The pharmaceutical composition for oral administration according to claim 2, wherein

6. R １ Alcohol HOR １ and the alcohol is a sugar selected from altrose, arabinose, dextrose, erythrose, fructose, galactose, glucose, gulose, idose, lactose, lyxose, mannose, ribose, ribulose, sucrose, talose, threose, and xylose.

7. The oligomer has the formula II 【Chemistry 2】 is a compound of the formula - R １ is as claimed in any one of claims 2 to 6, The pharmaceutical composition for oral administration according to claim 1, wherein n is an integer from 2 to 100.

8. formula 【Chemistry 3】 7. The pharmaceutical composition for oral administration according to claim 1, wherein the compound is (R)-3-hydroxybutyric acid (R)-1,3-butanediol monoester.

9. 9. A pharmaceutical composition for oral administration according to any one of claims 1 to 8 for use in treating a subject suffering from fatty liver.

10. The subject is １ 10. The pharmaceutical composition for oral administration of claim 9, having greater than 5% liver fat by weight as measured using H magnetic resonance spectroscopy (MRS).

11. 11. The pharmaceutical composition for oral administration according to any one of claims 1 to 10, wherein the pharmaceutical composition for oral administration is administered to a subject at least once daily, and the level of fat in the liver is reduced by at least 1 weight percentage point of the liver, preferably by at least 3 weight percentage points of the liver, and desirably by at least 5 weight percentage points of the liver, 5 days after the first treatment, compared to the level of fat in the liver before the first treatment.

12. 12. The pharmaceutical composition for oral administration according to any one of claims 1 to 11, wherein the disorder is fatty liver disease, preferably the disorder is non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), or non-alcoholic fatty liver (NAFL).

13. 13. A pharmaceutical composition for oral administration according to any one of claims 1 to 12 for use in treating a subject who is overweight (having a BMI in the range of 25 to less than 30), an obese subject (having a BMI in the range of 30 to less than 40), or a severely obese subject (having a BMI of 40 or greater).

14. 14. A pharmaceutical composition for oral administration according to any one of claims 1 to 13, wherein the subject is diabetic or prediabetic.

15. 12. A pharmaceutical composition for oral administration according to any one of claims 1 to 11 for use in reducing the level of fat in the liver of a healthy subject.

16. 16. A pharmaceutical composition for oral administration according to any one of claims 1 to 15, wherein the dosage administered to a subject is greater than 100 mg / kg per day, preferably greater than 300 mg / kg per day.

17. 17. A pharmaceutical composition for use in preventing or treating fatty liver disease by reducing liver fat, comprising a pharmaceutical composition for oral administration according to any one of claims 1 to 16 and one or more pharma- ceutically acceptable excipients.

18. 17. A nutritional composition for use in preventing or treating fatty liver disease by reducing liver fat comprising a pharmaceutical composition for oral administration according to any one of claims 1 to 16, and optionally further comprising water and one or more of flavourings, proteins, carbohydrates, sugars, fats, fibre, vitamins and minerals.

19. Further comprising a medium chain triglyceride, preferably the medium chain triglyceride has the formula CH ２ R ａ -CH ２ R ｂ -CH ２ R ｃ wherein R ａ , R ｂ , and R ｃ 20. The nutritional composition of claim 18, wherein is a fatty acid having from 5 to 12 carbon atoms.

20. Use of a pharmaceutical composition for oral administration according to any one of claims 1 to 8, or a composition according to any one of claims 17 to 19, in the manufacture of a medicament for use in preventing or treating fatty liver disorders by reducing liver fat in a subject, preferably according to any one of claims 9 to 16.

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