The present invention relates to a composition for use in treating and/or preventing a non-alcoholic fatty liver disease (NAFLD) in a subject in need thereof, wherein said composition comprises the following ingredients: Hepar suis, Cyanocobalaminum, Duodenum suis, Thymus suis, Colon suis, Vesica fellea suis, Pankreas suis, Cinchona pubescens, Lycopodium clavatum, Chelidonium majus, Silybum marianum, Histaminum, Sulfur, Avena sativa, Natrium diethyloxalaceticum, Acidum alpha-ketoglutaricum, Acidum malicum, Acidum fumaricum, Calcium carbonicum Hahnemanni, Taraxacum officinale, Cynara scolymus, Veratrum album, Acidum thiocticum, and Acidum oroticum monohydricum. The invention also relates to a pharmaceutical composition comprising said composition and at least one further drug against NAFLD. Moreover, the present invention also contemplates a kit for use in treating and/or preventing NAFLD in a subject in need thereof comprising the (i) said composition and (ii) at least one further drug against NAFLD.

NAFLD is an umbrella term for a spectrum of abnormalities which are defined by the presence of hepatic steatosis and observed in people who consume no or only limited amounts of alcohol (Lucas et al., 2018). It comprises a non-alcoholic fatty liver (NAFL) which is characterized by the presence of hepatic steatosis without evidence for substantial inflammation or hepatocyte ballooning and non-alcoholic steatohepatitis (NASH) which is defined by the presence of hepatic steatosis associated with inflammation, hepatocellular ballooning with and without fibrosis (Benedict & Zhang, 2017).

The global prevalence of NAFLD is around 25% and it is increasing continuously over years due to the obesity crises, the rise in diabetes and the metabolic syndrome (Iqbal et al., 2019). It has been projected to become the main cause of liver related morbidity and mortality within the next 20 years and it is rapidly becoming the leading indication for a liver transplantation (Benedict & Zhang, 2017). The growing NAFLD epidemic will constitute a significant burden for our health care system (Younossi, 2019).

Several risk factors have been suggested for the development of NAFLD including metabolic disorders such as the metabolic syndrome and type 2 diabetes, endocrine disorders, obstructive sleep apnea, different medications, lifestyle factors such as a poor diet (rich in glucose, fructose, and saturated fat), a sedentary behavior, smoking, age and others (Benedict & Zhang, 2017). The pathogenesis of NAFLD includes a complex interaction of multiple factors and can be influenced by a genetic predisposition (Yu et al., 2016). An unhealthy lifestyle accompanied by a poor diet with a positive energy balance and a sedentary behavior can lead to an increase in fat mass, insulin resistance, type 2 diabetes, gut dysbiosis and hepatic de novo lipogenesis (Stefan et al., 2019). Type 2 diabetes associated with high levels of insulin and glucose can exacerbate hepatic de novo lipogenesis (Stefan et al., 2019). Gut dysbiosis and leakiness can induce liver inflammation and fibrosis via an increased or dysregulated release of bacteria-derived products (Stefan et al., 2019). The pathophysiological changes in the liver can be enhanced by visceral obesity and a lipodystrophy-like phenotype (Stefan et al., 2019).

The diagnosis of NAFLD is based on four criteria, hepatic steatosis is present, detected via imaging or histology, no or only limited amounts of alcohol are consumed, there are no rival etiologies or other causes for a chronic liver disease (Benedict & Zhang, 2017).

So far there is no specific drug approved by regulatory agencies for the specific treatment of NAFL or NASH (Pydyn et al., 2020). This means currently prescribed drugs are used off-label and no specific pharmacotherapy can be recommend for this disease.

There is a need for compositions that can be used for treating and/or preventing hepatic diseases and, in particular, NAFLD.

The technical problem underlying the present invention can be seen as the provision of means and methods for complying with the aforementioned needs. The technical problem is solved by the embodiments characterized in the claims and herein below.

The present invention relates to a composition for use in treating and/or preventing a non-alcoholic fatty liver disease (NAFLD) in a subject in need thereof, wherein said composition comprises the following ingredients: Hepar suis, Cyanocobalaminum, Duodenum suis, Thymus suis, Colon suis, Vesica fellea suis, Pankreas suis, Cinchona pubescens, Lycopodium clavatum, Chelidonium majus, Silybum marianum, Histaminum, Sulfur, Avena sativa, Natrium diethyloxalaceticum, Acidum alpha-ketoglutaricum, Acidum malicum, Acidum fumaricum, Calcium carbonicum Hahnemanni, Taraxacum officinale, Cynara scolymus, Veratrum album, Acidum thiocticum, and Acidum oroticum monohydricum. The term "composition" as used herein refers to a mixture of extracts from, inter alia, biological sources such as animals, plants and parts thereof, such as organs, which are further defined elsewhere herein. Preferably, the said composition can comprise further ingredients.

Preferably envisaged in accordance with the present invention is a composition which comprises as one or more further ingredient(s) at least one pharmaceutically acceptable carrier and/or diluent. The pharmaceutically acceptable carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and being not deleterious to the recipient thereof and is selected so as not to affect the biological activity of the combination. The pharmaceutical carrier employed may include a solid, a semi-solid or a liquid. Exemplary of solid carriers are lactose, microcrystalline cellulose, starch, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, disintegrating agents, lubricants colouring matter, flavouring substances and the like. Similarly, the carrier or diluent may include time delay material (release controlling agents) well-known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax. Preferred carriers for semi-solid or liquid compositions encompass (distilled) water, alcohols, glycerol, physiological saline solutions, buffers, such as phosphate buffered saline solutions, Ringer's solutions, dextrose solution, and Hank's solution, syrup, oil, emulsions, various types of wetting agents, hard fat, macrogols, cocoa butter, thickeners such as carbomers, and the like. Said suitable carriers comprise those mentioned above, and others well-known in the art, see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, European Pharmacopeia, Homeopathic Pharmacopeia of the USA or HAB. In addition, the pharmaceutical composition or formulation may also include other carriers, stabilizing agents, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like. Furthermore, the formulation may include additional pharmaceutically active agents.

The composition shall be adapted for use in treating and/or preventing NAFLD. Accordingly, it will be understood that dependent on the desired mode of administration the composition shall be formulated for a systemic or topical application. Typically, systemic administration may be achieved by administration routes such as intra-muscular, subcutaneous, oral or intravenous. Preferably, the composition envisaged herein is formulated for a systemic application. Preferably, oral application, e.g. in the form of tablets, solution or drinking ampules, is envisaged or application via injection. The composition can be, preferably, formulated for a bolus administration or can be made for continuous applications as set forth elsewhere herein in detail. Preferably, the composition according to the present invention is formulated as a medicament as set forth elsewhere herein in detail.

The term “non-alcoholic fatty liver disease (NAFLD)” as used herein refers to a disease which is accompanied by pathological accumulation of fat in the liver as a result of e.g. increased supply of fat and excess adipose tissue lipolysis, decreased export of fat in form of very low density lipoproteins, decreased free fatty acid beta-oxidation and/or increased de novo lipogenesis (Yu et al., 2016). Typically, patients suffering from NAFLD do not significantly consume alcohol (Lucas et al., 2018). NAFLD refers to a continuous group of pathological liver disorders ranging from non-alcoholic fatty liver (NAFL, simple steatosis) to non-alcoholic steatohepatitis (NASH) (Benedict & Zhang, 2017). Common to these disorders is that they begin with fat accumulation in the liver (hepatic steatosis). A liver can remain fatty without disturbing liver function (NAFL), but by various mechanisms and possible insults to the liver, it may also progress into a non-alcoholic steatohepatitis (NASH) (Stefan et al., 2019). NAFLD as referred to herein may involve inflammation and, thus, encompasses forms of hepatitis and, in particular, non-alcoholic steatohepatitis (NASH). In NASH, steatosis is combined with inflammation and sometimes fibrosis (Benedict & Zhang, 2017). NASH can lead to complications such as cirrhosis and hepatocellular carcinoma (Benedict & Zhang, 2017). Preferably, said NAFLD referred to in accordance with the present invention encompasses NASH.

NAFLD patients may remain asymptomatic, however, some patients may complain of fatigue, malaise, and dull right-upper-quadrant abdominal discomfort (Lucas et al., 2018). Clinical and diagnostic parameters for the disease are well-known to the clinician and described in standard text books of medicine. NAFLD may cause symptoms related to liver dysfunction and can be diagnosed by performing imaging and/or a liver biopsy and after other potential causes of hepatic steatosis have been ruled (Benedict & Zhang, 2017). Indeed, in cases of symptoms or signs attributable to liver disease or when chemical tests show abnormal liver function, NAFLD should be suspected and investigated (Sharma & Arora, 2020). When no symptoms or signs attributable to liver disease are reported or when the tests show normal liver chemistries, but a hepatic steatosis is detected, NAFLD should be suspected and investigated as well (Sharma & Arora, 2020). Other metabolic risk factors (e.g., obesity, diabetes mellitus, dyslipidemia) and alternate causes such as alcohol should be taken into account for establishing a diagnosis (Sharma & Arora, 2020).

NAFLD may be associated with or caused by comorbidities including diabetes mellitus type II and metabolic syndromes, such as obesity, combined dyslipidemia (high triglyceride levels and low HDL-cholesterol levels), insulin resistance, and hypertension (Benedict & Zhang, 2017). It may be also associated with hormonal disorders, such as panhypopituitarism, hypothyroidism, hypogonadism, polycystic ovary syndrome, persistently elevated transaminases, increasing age and BMI and hypoxia caused by obstructive sleep apnea (Benedict & Zhang, 2017; Lonardo et al. 2019).

Preferably, physiological changes observed in subjects suffering from NAFLD or those being at risk therefor are increased average adipocyte size in mesenteric white adipose tissue, increased number of inflammatory aggregates in the liver, differential expression of genes in the liver, affected upstream regulator cholesterol in the liver, increased concentration of free cholesterol in the liver, increased concentration of cholesteryl ester in the liver, increased net cholesterol production, and/or increased number of neutrophil aggregates in the liver, increased liver weight, increased liver and plasma triglyceride levels, increased NAFLD activity score values, increased fibrosis area and collagen deposition in the liver as well as increased neutrophil numbers in the liver. Preferably, a subject suffering from NAFLD and, preferably, from NASH, shall exhibit one or more of the aforementioned physiological changes and more preferably, increased average adipocyte size in mesenteric white adipose tissue, increased number of inflammatory aggregates in the liver, increased concentration of free cholesterol in the liver, increased concentration of cholesteryl ester in the liver, and/or increased number of neutrophil aggregates in the liver, increased liver and plasma triglyceride levels, increased NAFLD activity score values, and increased fibrosis area and collagen deposition in the liver as well as increased neutrophil numbers in the liver. More preferably, said subject may exhibit increased average adipocyte size in mesenteric white adipose tissue, increased number of inflammatory aggregates in the liver, differential expression of genes in the liver, affected upstream regulator cholesterol in the liver, increased concentration of free cholesterol in the liver, increased concentration of cholesteryl ester in the liver, increased net cholesterol production, and/or increased number of neutrophil aggregates in the liver, increased liver weight, increased liver and plasma triglyceride levels, increased NAFLD activity score values, and increased fibrosis area and collagen deposition in the liver as well as increased neutrophil numbers in the liver. In subjects having epidi dymal white adipose tissue, there is, preferably, an increase in the number of crown-like structures.

The term "treating" as used herein refers to any improvement or amelioration of the disease or symptom thereof as referred to herein. However, more preferably, treating means improving NAFLD such that liver tissue morphology and/or liver function are significantly improved. It will be understood that treatment may not occur in 100% of the subjects to which the composition has been administered. The term, however, requires that the treatment occurs in a statistically significant portion of subjects (e.g. a cohort in a cohort study). Whether a portion is statistically significant can be determined without further ado by a person skilled in the art using various well- known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney-U test etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %. The p-values are, preferably, 0.05, 0.01, 0.005, 0.001, or 0.0001.

The term “preventing” as used herein refers to significantly reducing the likelihood with which the disease or the development of symptoms thereof develop in a subject within a defined window (prevention window) starting from the administration of the composition onwards. Typically, the prevention window is within 1 to 5 days, within 1 to 3 weeks, within 1 to 3 months. The prevention window depends on the amount of composition which is administered and the applied dosage regimen. Typically, suitable prevention windows can be determined by the clinician based on the amount of composition to be administered and the dosage regimen to be applied without further ado. It will be understood that prevention may not occur in 100% of the subjects to which the composition has been administered. The term, however, requires that the prevention occurs in a statistically significant portion of subjects (e.g. a cohort in a cohort study). Whether a portion is statistically significant can be determined without further ado by a person skilled in the art using various well-known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney-U test etc. Details are described elsewhere herein.

Preferably, the composition of the present invention upon administration to the subject suffering from NAFLD or being at risk therefor causes at least one physiological effect selected from the group consisting of:

• reduces the average adipocyte size in mesenteric white adipose tissue,

• reduces the number of crown-like structures in epididymal white adipose tissue,

• reduces the number of inflammatory aggregates in the liver,

• differentially expressed genes in the liver, preferably 50 different genes,

• affects the upstream regulator cholesterol in the liver,

• reduces the concentration of free cholesterol in the liver,

• reduces the concentration of cholesteryl ester in the liver,

• reduces the net cholesterol production,

• reduces the number of neutrophil aggregates in the liver (GR-1 -positive cell aggregates),

• reduces liver weight,

• reduces liver triglycerides

• reduces plasma triglycerides,

• reduces NAFLD activity score values,

• reduces liver fibrosis (Sirius red area),

• reduces collagen deposition in the liver, and/or

• reduces neutrophil numbers in the liver (GR-1 -positive cells).

The term “subject” as used herein refers to a mammal and preferably a human. Said subject shall be, preferably, in need of treatment and/or prevention of NAFLD, i.e. it shall exhibit the disease or be at risk therefor. More preferably, a subject being at risk for NAFLD suffers from one or more of the comorbidities of NAFLD mentioned above and, in particular, from diabetes mellitus type II and metabolic syndromes, such as obesity, combined dyslipidemia (high triglyceride levels and low HDL-cholesterol levels), insulin resistance, hypertension, hormonal disorders, such as panhypopituitarism, hypothyroidism, hypogonadism, polycystic ovary syndrome, persistently elevated transaminases, increasing age and BMI and/or hypoxia caused by obstructive sleep apnea (Benedict & Zhang, 2017). Further risk factors for NAFLD comprise Hispanic ethnical background, smoking, personal fitness and sedentary behavior, nutritional diets, in particular, high consumption of red meat, refined grains, pastries, and/or sugar laden beverages, medication, in particular, administration of amiodarone, nucleoside analogues, aspirin, oestrogens, glucocorticosteroids, methotrexate, tamoxifen and/or tetracycline, total parenteral nutrition, severe anemia, inflammatory bowel disease, inborn errors of metabolism, and/or intake of barium salts, chromates, thallium, and phosphorus (Benedict & Zhang, 2017; Lucas et al., 2018).

The plants referred to in accordance with the present invention, i.e. Cinchona pubescens (cinchona bark), Lycopodium clavatum (club moss), Chelidonium majus (greater celandine), Silybum marianum (milk thistle), Avena sativa (oat), Taraxacum officinale (common dandelion), Cynara scolymus (globe artichoke), and Veratrum album (white hellebore), are well-known to the person skilled in the art and botanically characterized. Accordingly, the said plants as a starting material for preparing the composition according to the present invention can be identified, cultured and/or harvested without further ado. For example, the plants can be obtained by growing developing seedlings in the green house under standard conditions of humidity, temperature, and illumination into plants, culturing said plants for a period of time sufficient to allow for production of secondary plant metabolites and harvesting the plants or parts thereof required for the extraction process defined elsewhere herein. Furthermore, the said plants as a starting material for preparing the composition according to the present invention can, preferably, also be obtained from wild harvesting. In this case, the plants are identified using micro- and macroscopic testing methods.

Other components of the composition are also well-known chemicals in homeopathic applications, such as Cyanocobalaminum (cyanocobalamin), Histaminum (histamine), Sulfur, Natrium diethyloxalaceticum (sodium diethyl oxalacetate), Acidum alpha-ketoglutaricum (alphaketoglutaric acid), Acidum malicum (malic acid), Acidum fumaricum (fumaric acid), Calcium carbonicum Hahnemanni (inner parts of broken shells of the oyster), Acidum thiocticum (lipoic acid), and Acidum oroticum monohydricum (orotic acid monohydrate). Again, these components, in particular as homeopathic ingredients, are well-known to the person skilled in the art.

Yet further components of the composition are also well-known in homeopathic applications and are extracts from animal organs or tissues. In particular, components from pigs, such as Hepar suis (liver), Duodenum suis (duodenum), Thymus suis (thymus), Colon suis (colon), Vesica fellea suis (gall bladder), and Pankreas suis (pancreas) are also well-known to the person skilled in the art. Moreover, the pigs (Sus scrofa domesticus) used for obtaining the aforementioned tissue and organs are kept under controlled conditions for homeopathic purposes.

The composition according to the present invention comprises the said components in the following homeopathic dilutions: Hepar suis D8, Cyanocobalaminum D4, Duodenum suis DIO, Thymus suis DIO, Colon suis DIO, Vesica fellea suis DIO, Pankreas suis DIO, Cinchona pubescens D4, Lycopodium clavatum D4, Chelidonium majus D5, Silybum marianum D3, Histaminum DIO, Sulfur D13, Avena sativa D6, Natrium diethyloxalaceticum DIO, Acidum alpha-ketoglutaricum DIO, Acidum malicum DIO, Acidum fumaricum DIO, Calcium carbonicum Hahnemanni D28, Taraxacum officinale D4, Cynara scolymus D6, Veratrum album D4, Acidum thiocticum D8, and Acidum oroticum monohydricum D6.

The term “Hepar suis D8“ as used herein refers to a dilution of Hepar suis mother tincture (corresponding to the dilution DI) which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, as used herein HAB may refer to HAB according to 2019 and Ph. Eur. to version 10.1. Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. D-Dilutions of the mother tincture can be made by applying the following dilution scheme: One part of mother tincture (= dilution DI) is diluted in 9 parts glycerol 85 % to obtain a D2 dilution. One part of this dilution D2 is then diluted in 9 parts ethanol 18.5 % (V/V) to obtain a dilution D3. The dilutions D4 up to D6 are produced accordingly. The dilution D6 is further processed as described below.

The term “Cyanocobalaminum D4“ as used herein refers to a dilution of Cyanocobalaminum solution D2 which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, the aforementioned solution to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. The solution D2 is further processed as described below.

The term “Duodenum suis D10“ as used herein refers to a dilution of Duodenum suis mother tincture (corresponding to the dilution DI) which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, the aforementioned mother tincture (corresponding to the dilution DI) to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. One part of mother tincture (= dilution DI) is diluted in 9 parts glycerol 85 % to obtain a dilution D2. One part of this dilution D2 is then diluted in 9 parts ethanol 18.5 % (V/V) to obtain a dilution D3. The dilutions D4 up to D8 are produced accordingly. The dilution D8 is further processed as described below. The term “Thymus suis D10“ as used herein refers to a dilution of Thymus suis mother tincture (corresponding to the dilution DI) which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. One part of mother tincture (= dilution DI) is diluted in 9 parts glycerol 85 % to obtain a dilution D2. One part of this dilution D2 is then diluted in 9 parts ethanol 18.5 % (V/V) to obtain a dilution D3. The dilutions D4 up to D8 are produced accordingly. The dilution D8 is further processed as described below.

The term “Colon suis D10“ as used herein refers to a dilution of Colon suis mother tincture (corresponding to the dilution DI) which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. One part of mother tincture (= dilution DI) is diluted in 9 parts glycerol 85 % to obtain a dilution D2. One part of this dilution D2 is then diluted in 9 parts ethanol 18.5 % (V/V) to obtain a dilution D3. The dilutions D4 up to D8 are produced accordingly. The dilution D8 is further processed as described below.

The term “Vesica fellea suis D10“ as used herein refers to a dilution of Vesica fellea suis mother tincture (corresponding to the dilution DI) which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. One part of mother tincture (= dilution DI) is diluted in 9 parts glycerol 85 % to obtain a dilution D2. One part of this dilution D2 is then diluted in 9 parts ethanol 18.5 % (V/V) to obtain a dilution D3. The dilutions D4 up to D8 are produced accordingly. The dilution D8 is further processed as described below.

The term “Pankreas suis D10“ as used herein refers to a dilution of Pankreas suis mother tincture (corresponding to the dilution DI) which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. One part of mother tincture (= dilution DI) is diluted in 9 parts glycerol 85 % to obtain a dilution D2. One part of this dilution D2 is then diluted in 9 parts ethanol 18.5 % (V/V) to obtain a dilution D3. The dilutions D4 up to D8 are produced accordingly. The dilution D8 is further processed as described below. The term “Cinchona pubescens D4“ as used herein refers to a dilution of Cinchona pubescens mother tincture (corresponding to the dilution DI) which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. One part of mother tincture is diluted in 9 parts ethanol (70 % V/V) to obtain a dilution D2. This dilution is further processed as described below.

The term “Lycopodium clavatum D4“ as used herein refers to a dilution of Lycopodium clavatum mother tincture (corresponding to the dilution DI) which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur). Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. One part of mother tincture is diluted in 9 parts ethanol (90 % V/V) to obtain a dilution D2. This dilution is further processed as described below.

The term “Chelidonium majus D5“ as used herein refers to a dilution of Chelidonium majus mother tincture which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. Three parts of mother tincture are diluted in 7 parts ethanol (70 % V/V) to obtain a dilution DI. One part of this dilution DI is then diluted in 9 parts ethanol (70 % V/V) to obtain a dilution D2. The dilution D3 is produced accordingly. This dilution is further processed as described below.

The term “Silybum marianum D3“ as used herein refers to a dilution of Silybum marianum mother tincture which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. Three parts of mother tincture are diluted in 7 parts ethanol (50 % V/V) to obtain a dilution DI. This dilution is further processed as described below.

The term “Histaminum D10“ as used herein refers to a dilution of Histaminum solution DI which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, the aforementioned solution to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. One part of the solution DI is diluted in 9 parts ethanol 50 % (V/V) to obtain a dilution D2. The dilutions D3 up to D8 are produced accordingly. The dilution D8 is further processed as described below.

The term “Sulfur D13“ as used herein refers to a dilution of Sulfur solution D4 which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, the aforementioned solution to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. One part of the solution is diluted in 9 parts ethanol (90 % V/V) to obtain a D5 dilution. One part of the D5 dilution is diluted in 9 parts ethanol (70 % V/V) to obtain a D6 dilution. One part of the D6 dilution is diluted in 9 parts ethanol (50 % V/V) to obtain a D7 dilution. The dilutions D8 up to Dl l are produced accordingly. The dilution DI 1 is further processed as described below.

The term “Avena sativa D6“ as used herein refers to a dilution of Avena sativa mother tincture which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. Two parts of mother tincture are diluted in 8 parts ethanol (50 % V/V) to obtain a dilution DI . One part of the dilution DI is diluted in 9 parts ethanol 50 % (V/V) to obtain a dilution D2. The dilutions D3 up to D4 are produced accordingly. The dilution D4 is further processed as described below.

The term “Natrium diethyloxalaceticum D10“ as used herein refers to a dilution of Natrium diethyloxalaceticum trituration DI which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, the aforementioned trituration to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. One part of the trituration DI is diluted in 9 parts lactose monohydrate to obtain a trituration D2. The triturations D3 up to D6 are produced accordingly. One part of the trituration D6 is dissolved in 9 parts purified water and succussed to obtain a dilution D7. One part of the dilution D7 is diluted in 9 parts ethanol 36,2 % (V/V) to obtain a dilution D8. This dilution is further processed as described below.

The term “Acidum alpha-ketoglutaricum D10“ as used herein refers to a dilution of Acidum alpha- ketoglutaricum solution DI which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, the aforementioned solution to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. One part of the dilution DI is diluted in 9 parts ethanol 50 % (V/V) to obtain a dilution D2. The dilutions D3 up to D8 are produced accordingly. The dilution D8 is further processed as described below.

The term “Acidum malicum D10“ as used herein refers to a dilution of Acidum malicum solution DI which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, the aforementioned solution to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. One part of the dilution DI is diluted in 9 parts ethanol 50 % (V/V) to obtain a dilution D2. The dilutions D3 up to D8 are produced accordingly. The dilution D8 is further processed as described below.

The term “Acidum fumaricum D10“ as used herein refers to a dilution of Acidum fumaricum solution D2 which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, the aforementioned solution to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. One part of the dilution D2 is diluted in 9 parts ethanol 50 % (V/V) to obtain a dilution D3. The dilutions D4 up to D8 are produced accordingly. The dilution D8 is further processed as described below.

The term “Calcium carbonicum Hahnemanni D28“ as used herein refers to a dilution of Calcium carbonicum Hahnemanni trituration DI which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, the aforementioned trituration to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. One part of the trituration DI is diluted in 9 parts lactose monohydrate to obtain a trituration D2. The triturations D3 up to D6 are produced accordingly. One part of the trituration D6 is dissolved in 9 parts purified water and succussed to obtain a dilution D7. One part of the dilution D7 is diluted in 9 parts ethanol 36,2 % (V/V) to obtain a dilution D8. One part of the dilution D8 is diluted in 9 parts ethanol 50 % (V/V) to obtain a dilution D9. The dilutions D10 up to D26 are produced accordingly. The dilution D26 is further processed as described below.

The term “Taraxacum officinale D4“ as used herein refers to a dilution of Taraxacum officinale mother tincture which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. Two parts of the mother tincture are diluted in 8 parts ethanol 50 % (V/V) to obtain a dilution DI. One part of the dilution DI is diluted in 9 parts ethanol 50 % (V/V) to obtain a dilution D2. This dilution is further processed as described below.

The term “Cynara scolymus D6“ as used herein refers to a dilution of Cynara scolymus mother tincture which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. Three parts of mother tincture are diluted in 7 parts ethanol (70 % V/V) to obtain a dilution DI. One part of this dilution DI is then diluted in 9 parts ethanol (70 % V/V) to obtain a dilution D2. The dilution D3 is produced accordingly. One part of this dilution D3 is then diluted in 9 parts ethanol (50 % V/V) to obtain a dilution D4. This dilution is further processed as described below.

The term “Veratrum album D4“ as used herein refers to a dilution of Veratrum album mother tincture (corresponding to the dilution DI) which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. One part of mother tincture is diluted in 9 parts ethanol (70 % V/V) to obtain a dilution D2. This dilution is further processed as described below.

The term “Acidum thiocticum D8“ as used herein refers to a dilution of Acidum thiocticum trituration DI which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, the aforementioned trituration to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. One part of the trituration DI is diluted in 9 parts lactose monohydrate to obtain a trituration D2. The triturations D3 up to D6 are produced accordingly. The trituration D6 is further processed as described below.

The term “Acidum oroticum monohydricum D6“ as used herein refers to a dilution of Acidum oroticum monohydricum trituration DI which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB) or the European Pharmacopeia (Ph. Eur.). Preferably, the aforementioned trituration to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. One part of the trituration DI is diluted in 9 parts lactose monohydrate to obtain a trituration D2. The triturations D3 up to D4 are produced accordingly. The trituration D4 is further processed as described below.

The finally envisaged solutions or dilutions Hepar suis D6, Cyanocobalaminum D2, Duodenum suis D8, Thymus suis D8, Colon suis D8, Vesica fellea suis D8, Pankreas suis D8, Cinchona pubescens D2, Lycopodium clavatum D2, Chelidonium majus D3, Silybum marianum DI, Histaminum D8, Sulfur Dl l, Avena sativa D4, Natrium diethyloxalaceticum D8, Acidum alpha- ketoglutaricum D8, Acidum malicum D8, Acidum fumaricum D8, Calcium carbonicum Hahnemanni D26, Taraxacum officinale D2, Cynara scolymus D4, Veratrum album D2 as described above are subsequently mixed with each other and potentised twice with water for injections. The triturations (Acidum thiocticum D6, Acidum oroticum monohydricum D4) are mixed, then dissolved in water for injections and potentised, followed by a second potentisation step with water for injections.

Both final mixtures are mixed with water for injections. The sodium chloride is added and dissolved by mixing to obtain the composition to be used in accordance with the present invention.

Preferably, a composition envisaged for use in accordance with the present invention is the commercially available drug composition Hepar comp, as injection solution (Biologische Heilmittel Heel GmbH, Germany).

In the following, the components and amounts present in the composition for use according to the invention are listed for the injection solution (ampoules):

Typically, the composition for use according to the present invention is to be administered in an amount sufficient for treating and/or preventing NAFLD. It will be understood that the dosage to be applied may depend on several factors well-known to the clinician including gender, age, body weight and general health condition. Moreover, the dosage will depend on the kind of administration. Typically, a topical administration may require a different dosage from systemic administration. Similarly, the mode of administration will also affect the therapeutically effective dosage. For example, oral administration may require another dosage than intraperitoneal injection. Preferably, the composition according to the present invention shall be administered as a solution. More preferably, the dosage provided to a subject shall correspond to the amount provided by at least about 1.5 ml composition per kg body weight in mice administered 3 times a week for at least 18 weeks. More preferably, the dosage provided to a subject shall correspond to the amount provided by at least about 1.5 ml composition per kg body weight in mice administered every other day for at least 3 weeks.

If the composition for use according to the present invention is formulated as a solution, a 2.2 ml ampoule shall be administered per day (44 pl/kg body weight, considering an adult with 50 kg body weight). Such an ampoule may be administered between one to three times a week.

Preferably, the aforementioned dosage can be provided by a single administration (bolus) or may be achieved by more than one administration step within the day. Moreover, it is also envisaged that the dosage is, preferably, provided by using continuous release devices which provide for a continuous administration of the dosage over the day.

How the composition can be formulated in a proper manner in order to provide the aforementioned daily dosage is well-known in the art. Typical formulations may be tablets, soft and hard capsules, powders, pellets, granules (incl. effervescent granules), solutions, suspensions or emulsions such as tinctures for dermal or liquids for oral applications, injection or infusion solutions, creams, gels or ointments, suppositories or preparations for oromucosal use.

The composition according to the present invention may be used together with at least one further drug against NAFLD. Preferably, said further NAFLD drug may be an AOC3 inhibitor, CCR2/CCR5 receptor antagonist, PPAR alpha/delta agonist, caspase inhibitor, acetyl-CoA carboxylase inhibitor, FXR agonist, PPAR gamma agonist, ASK1 inhibitor, GLP-1 receptor agonist, SGLT2 inhibitor, THR-P agonist, SCD1 modulator, FXR agonist, FGF 19 analogue, THR-P agonist, FGF21 analogue, and/or DPP -4 inhibitor. The following drugs from the aforementioned drug groups have been shown to be, inter alia, suitable as drugs against NAFLD and are thus envisaged in accordance with the present invention, Cenicriviroc, Elafibranor, Emricasan, IVA337, GS0976, GS9674, Obeticholic acid, Pioglitazone, Selonsertib, Semaglutide, Liraglutide, Empagliflozin, Resmetirom, Norursodeoxycholic acid (side chain- shortened homologue of ursodeoxycholic acid), Aramchol, Tropifexor, NGM282, VK2809, Pegbelfermin:, Sitagliptin, Silymarin, Omega-3 fatty acids, Aspirin, and/or Atorvastatin. Besides these drugs, other homeopathic drugs or compositions, probiotics, prebiotics, herbal drugs, and/or vitamins, such as vitamin E, may also be applied together with the composition for use in accordance with the present invention.

The aforementioned further drugs against NAFLD may be administered together with the composition according to the invention or sequentially before or after administration of said composition. If the composition is to be administered together with the said at least one further drug, this may be achieved by administering a single composition consisting of the composition for use in accordance with the present invention and said at least one further drug or they may be administered as separate compositions.

Advantageously, it has been found in accordance with the present invention that a composition comprising the aforementioned ingredients when administered to mice suffering from high fat diet-induced NASH development was able to ameliorate NAFLD and, in particular, NASH. In diseased mice, the composition positively influenced several physiological changes compared to placebo treated animals. In particular, treated mice showed a reduced number of crown-like structures in epididymal white adipose tissue, a reduced average adipocyte size in mesenteric white adipose tissue, a reduced number of inflammatory aggregates in the liver, 50 differentially expressed genes in the liver, an affected upstream regulator cholesterol in the liver, reduced concentration of free cholesterol in the liver, reduced concentration of cholesteryl ester in the liver, reduced net cholesterol production, and reduced number of neutrophil aggregates in the liver (GR- 1 -positive cell aggregates). In another study, mice treated by the composition according to the present invention showed reduced liver weight, reduced plasma triglycerides, reduced liver triglycerides, reduced NAFLD activity score values, reduced liver fibrosis (Sirius red area), reduced collagen deposition in the liver, and/or reduced neutrophil numbers in the liver (GR-1- positive cells).

In particular, in one study underlying the present invention, the effect of Hepar comp. (HE-700) was evaluated in a preventive study protocol (starting treatment from t=0 weeks) and an early- therapeutic study protocol (starting treatment from t=6 weeks) on the development of nonalcoholic steatohepatitis (NASH) and metabolic risk factors in Ldlr-/-. Leiden mice fed a high fat diet. HE-700 was administered by intraperitoneal injection 3 times weekly and the mice were treated up to a period of 24 weeks (the end of the study). There was a corresponding vehicle control group for each treatment group (HE-900, started at either t=0 or t=6 weeks). HE-900 vehicle control injections and HE-700 injections were well tolerated, and all mice displayed normal behavior (no abnormalities were observed). Histological analysis of the adipose tissue showed that a preventative HE-700 treatment can significantly reduce the average adipocyte size in mesenteric white adipose tissue and significantly reduce the inflammation of epididymal white adipose tissue. Histopathological analysis of hepatic inflammation revealed that HE-700, when used in an early- therapeutic treatment protocol, can significantly reduce hepatic inflammation. The hepatic transcriptomics analysis revealed that HE-700 treatment downregulated Abcg8, a transporter for cholesterol excretion into the bile (Wang et al., 2015). The expression of this gene is upregulated under control of the transcription factor LXR when sterols accumulate in the liver (Wang et al., 2015). For instance, Abcg8 is upregulated when a lot of cholesterol is present in the liver, such as in mice fed a cholesterol-containing HFD (Kamisako et al., 2014). In line with this, Abcg8 was upregulated in HFD+HE-900 mice relative to chow controls. The observed downregulation in HE- 700-treated mice suggests that accumulation of cholesterol and/or bile acids in the liver may be reduced in HE-700-treated mice. In line with this, the upstream regulator analysis revealed that HE-700 treatment affected cholesterol as an upstream regulator (i.e. the gene expression pattern observed was in line with what would be expected when little cholesterol is present). Since cholesterol (specifically in its free/non-esterified form) is cytotoxic and a potent inducer of hepatic inflammation and hepatic fibrosis (loannou, 2016; Marra & Svegliati-Baroni, 2018), the suggested reduction in intrahepatic cholesterol levels could have contributed to the observed antiinflammatory effects. Further investigation of intrahepatic cholesterol content by biochemical analysis of liver lipids confirmed the observed effects on the gene expression level, showing a tendency towards reduced hepatic cholesteryl esters and a significant reduction in free cholesterol in HE-700-treated animals. This reduction in hepatic cholesterol accumulation provides a potential rationale for the observed anti-inflammatory effects of HE-700. To further substantiate the observed effect on intrahepatic cholesterol, the cholesterol balance (net cholesterol production = cholesterol excretion via neutral sterols and bile acids - dietary cholesterol intake) was calculated. The net cholesterol production was significantly reduced in animals treated with HE-700 and this is in line with the observed effects on intrahepatic cholesterol accumulation. The hepatic transcriptomics analysis further revealed that HE-700 treatment downregulated the neutrophil chemokine Cxcll which suggests that infiltration of neutrophilic cells may be reduced by HE-700. To investigate whether this is indeed the case, immunohistological analyses were performed and these confirmed the observed effects on the gene expression level, showing a significant reduction in the number of neutrophil aggregates (GR-1 -positive cell aggregates) in animals treated with HE-700. This observation further supports the anti-inflammatory nature of HE-700.

In yet another study underling the present invention, the effect of Hepar comp. (HE-700) was studied on the development of NAFLD in STAM mice (mice injected with streptozotocin shortly after birth and feeding of a high fat diet). HE-700 was administered by intraperitoneal injection every other day and the mice were treated for 3 or 4 weeks (till the end of the study). There was a corresponding vehicle control group for each treatment group (HE-900). It was found that NAS and the Sirius red-positive area (fibrosis area) were significantly increased in the NASH mice compared with the controls. HE-700 significantly reduced the fibrosis area and NAS in both cohorts (5-9 weeks, 6-9 weeks) compared with the corresponding vehicle group, again demonstrating an anti-NASH and an anti-fibrotic effect of HE-700. Notably, earlier treatment of HE-700 tended to reduce the liver triglyceride content, suggesting that the favorable effect of HE- 700 on the lipid metabolism became obvious by earlier intervention. However, a trend towards reduced liver weight and plasma triglyceride levels was only observed with the late intervention. Furthermore, HE-700 reduced hepatocellular ballooning, lobular inflammation and the Gr-1- positive cells in the liver compared with the vehicle group. These results indicated that HE-700 had anti-inflammatory effects in this model. Reduced signals of type 1 and 3 collagens were observed in HE-700-treated groups further supporting the anti-fibrotic effect of HE-700. Together, HE-700 had an anti-fibrotic, anti-inflammatory and hepatoprotective effect on NASH in this study.

Further details are described in the accompanying examples below. Thanks to the present invention it will be possible to more effectively treat and/or prevent NAFLD.

In general, the present invention also relates to a method of treating and/or preventing NAFLD in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a composition comprising the following ingredients: Hepar suis, Cyanocobalaminum, Duodenum suis, Thymus suis, Colon suis, Vesica fellea suis, Pankreas suis, Cinchona pubescens, Lycopodium clavatum, Chelidonium majus, Silybum marianum, Histaminum, Sulfur, Avena sativa, Natrium diethyloxalaceticum, Acidum alpha-ketoglutaricum, Acidum malicum, Acidum fumaricum, Calcium carbonicum Hahnemanni, Taraxacum officinale, Cynara scolymus, Veratrum album, Acidum thiocticum, and Acidum oroticum monohydricum. Typically, a subject being in need of treatment and/or prevention of NAFLD is a subject as specified elsewhere herein, and, in particular a subject suffering from a comorbidity of NAFLD or being at risk therefor as mentioned herein.

In the following, particular preferred embodiments of the composition for use in accordance with the present invention are specified:

In a preferred embodiment of the composition for use of the invention said ingredients of the composition are used in the following homeopathic dilutions: Hepar suis D8, Cyanocobalaminum D4, Duodenum suis DIO, Thymus suis DIO, Colon suis DIO, Vesica fellea suis DIO, Pankreas suis DIO, Cinchona pubescens D4, Lycopodium clavatum D4, Chelidonium majus D5, Silybum marianum D3, Histaminum DIO, Sulfur D 13, Avena sativa D6, Natrium diethyloxalaceticum DIO, Acidum alpha-ketoglutaricum DIO, Acidum malicum DIO, Acidum fumaricum DIO, Calcium carbonicum Hahnemanni D28, Taraxacum officinale D4, Cynara scolymus D6, Veratrum album D4, Acidum thiocticum D8, and Acidum oroticum monohydricum D6.

In a preferred embodiment of the composition for use of the present invention, said composition is to be administered in an amount corresponding to the amount provided by at least about 1.5 ml composition per kg body weight in mice administered 3 times a week for at least 18 weeks. More preferably, said composition upon administration to the subject suffering from NAFLD or being at risk therefor reduced the number of crown-like structures in epididymal white adipose tissue, reduced the average adipocyte size in mesenteric white adipose tissue, reduced the number of inflammatory aggregates in the liver, led to a differential expression of genes in the liver, affected the upstream regulator cholesterol in the liver, reduced the concentration of free cholesterol in the liver, reduced the concentration of cholesteryl ester in the liver, reduced the net cholesterol production, and/or reduced the number of neutrophil aggregates in the liver (GR-1 -positive cell aggregates).

In yet a preferred embodiment of the composition for use of the present invention, said composition is to be administered in an amount corresponding to the amount provided by at least about 1.5 ml composition per kg body weight in mice administered every other day for at least 3 weeks. More preferably, said composition upon administration to the subject suffering from NAFLD or being at risk therefor reduced liver weight, reduced plasma triglycerides, reduced liver triglycerides, reduced NAFLD activity score values, reduced liver fibrosis (Sirius red area), reduced collagen deposition in the liver, and/or reduced neutrophil numbers in the liver (GR-1- positive cells).

In a preferred embodiment of the composition for use of the invention, said composition is to be administered to the subject together with at least one further drug against NAFLD. In a more preferred embodiment of the said composition for use, said at least one further drug against NAFLD is an AOC3 inhibitor, CCR2/CCR5 receptor antagonist, PPAR alpha/delta agonist, caspase inhibitor, acetyl-CoA carboxylase inhibitor, FXR agonist, PPAR gamma agonist, ASK1 inhibitor, GLP-1 receptor agonist, SGLT2 inhibitor, THR-P agonist, SCD1 modulator, FXR agonist, FGF 19 analogue, THR-P agonist, FGF21 analogue, and/or DPP-4 inhibitor. In yet a preferred embodiment of the said composition for use, said at least one further drug against NAFLD is selected from the group consisting of: Cenicriviroc, Elafibranor, Emricasan, IVA337, GS0976, GS9674, Obeticholic acid, Pioglitazone, Selonsertib, Semaglutide, Liraglutide, Empagliflozin, Resmetirom, Norursodeoxycholic acid (side chain- shortened homologue of ursodeoxycholic acid), Aramchol, Tropifexor, NGM282, VK2809, Pegbelfermin:, Sitagliptin, Silymarin, Omega-3 fatty acids, Aspirin, and/or Atorvastatin.

In a preferred embodiment of the composition for use of the invention said subject is a human.

The present invention also relates to a pharmaceutical composition comprising the composition according to the present invention and at least one further drug against NAFLD, preferably, as described elsewhere herein. Preferably, said at least one further drug against NAFLD is an AOC3 inhibitor, CCR2/CCR5 receptor antagonist, PPAR alpha/delta agonist, caspase inhibitor, acetyl- CoA carboxylase inhibitor, FXR agonist, PPAR gamma agonist, ASK1 inhibitor, GLP-1 receptor agonist, SGLT2 inhibitor, THR-P agonist, SCD1 modulator, FXR agonist, FGF 19 analogue, THR-P agonist, FGF21 analogue, and/or DPP-4 inhibitor. Also preferably, said at least one further drug against NAFLD may be selected from the group consisting of Cenicriviroc, Elafibranor, Emricasan, IVA337, GS0976, GS9674, Obeticholic acid, Pioglitazone, Selonsertib, Semaglutide, Liraglutide, Empagliflozin, Resmetirom, Norursodeoxycholic acid (side chain- shortened homologue of ursodeoxycholic acid), Aramchol, Tropifexor, NGM282, VK2809, Pegbelfermin:, Sitagliptin, Silymarin, Omega-3 fatty acids, Aspirin, and/or Atorvastatin.

The present invention also relates to a kit for use in treating and/or preventing NAFLD in a subject in need thereof comprising the (i) composition according to the present invention and (ii) at least one further drug against NAFLD, preferably, as described elsewhere herein.

A “kit” as referred to herein refers to a collection of items comprising the composition according to the present invention as well as at least one further drug against NAFLD which are provided together in a single container or separate containers in a “ready-to-use” form. Preferably, said kit also comprises instructions for use, i.e. instructions for proper administration of the drugs and/or for preparation.

All references cited throughout this description are herewith incorporated by reference with respect to their disclosure contents in their entireties and with respect to the disclosure content to which it is specifically referenced.

FIGURES

Figure 1: Effects of HE-700 treatment on the average adipocyte size of the mesenteric white adipose tissue (mW AT) in HFD-fed Ldlr-/-. Leiden mice. HE-700 treatment and HE-900 vehicle control treatment (both 1.5 ml/kg body weight, 3 times per week) were started att=O weeks (early) or t=6 weeks (late) and continued until the end of the study (t=24 weeks). Chow-fed animals were included as a reference and are shown in both the early-treatment and late-treatment graphs. A) Average adipocyte size of mW AT in mice treated from t=0 weeks. B) Average adipocyte size of mW AT in mice treated from t=6 weeks. * p<0.05, *** p<0.001 vs. respective HFD+HE-900 control.

Figure 2: Effects of HE-700 treatment on epididymal white adipose tissue (eWAT) inflammation in HFD-fed Ldlr-/-. Leiden mice. HE-700 treatment and HE-900 vehicle control treatment (both 1.5 ml/kg body weight, 3 times per week) were started at t=0 weeks (early) or t=6 weeks (late) and continued until the end of the study (t=24 weeks). Chow-fed animals were included as a reference and are shown in both the early-treatment and late-treatment graphs. A) eWAT inflammation in mice treated from t=0 weeks. B) eWAT inflammation in mice treated from t=6 weeks. * p<0.05, *** p<0.001 vs. respective HFD+HE-900 control.

Figure 3: Effects of HE-700 treatment on hepatic inflammation in HFD-fed Ldlr-/-. Leiden mice. HE-700 treatment and HE-900 vehicle control treatment (both 1.5 ml/kg body weight, 3 times per week) were started att=O weeks (early) ort=6 weeks (late) and continued until the end of the study (t=24 weeks). Chow-fed animals were included as a reference and are shown in both the early- treatment and late-treatment graphs. A) Hepatic inflammation in mice treated from t=0 weeks. B) Hepatic inflammation in mice treated from t=6 weeks. * p<0.05, *** p<0.001 vs. respective HFD+HE-900 control.

Figure 4: Effects of HE-700 treatment on hepatic neutrophil aggregates in HFD-fed Ldlr-/-. Leiden mice. HE-700 treatment and HE-900 vehicle control treatment (both 1.5 ml/kg body weight, 3 times per week) were started at t=6 weeks (late) and continued until the end of the study (t=24 weeks). Chow-fed animals were included as a reference. A) Hepatic neutrophil aggregates in mice treated from t=6 weeks displayed as graph. B) Hepatic neutrophil aggregates in mice treated from t=6 weeks displayed as representative microscopic images (aggregates are indicated with a black arrow). ** p<0.01, *** p<0.001 vs. respective HFD+HE-900 control.

Figure 5: Effects of HE-700 treatment on liver lipids in HFD-fed Ldlr-/-. Leiden mice. HE-700 treatment and HE-900 vehicle control treatment (both 1.5 ml/kg body weight, 3 times per week) were started at t=6 weeks (late) and continued until the end of the study (t=24 weeks). Chow-fed animals were included as a reference. A) Intrahepatic triglyceride levels in mice treated from t=6 weeks. B) Intrahepatic cholesteryl ester levels in mice treated from t=6 weeks. C) Intrahepatic free (non-esterified) cholesterol levels in mice treated from t=6 weeks. D) Correlation of free cholesterol and inflammatory aggregates in the liver. p=0.057, ** p<0.01, *** p<0.001 vs. respective HFD+HE-900 control.

Figure 6: Effects of HE-700 treatment on net cholesterol production in HFD-fed Ldlr-/-. Leiden mice. HE-700 treatment and HE-900 vehicle control treatment (both 1.5 ml/kg body weight, 3 times per week) were started at t=6 weeks (late) and continued until the end of the study (t=24 weeks). A) Net cholesterol production in mice at t=16 weeks. B) Net cholesterol production in mice at t=24 weeks. p=0.06, ** p<0.01 vs. HFD+HE-900 control.

Figure 7: Effects of HE-700 treatment on liver weight in STAM mice (STZ injection and HFD- feeding). HE-700 treatment and HE-900 vehicle control treatment (both 1.5 ml/kg body weight, every other day) were started at t=5 weeks or t=6 weeks and continued until the end of the study (t=9 weeks). Chow-fed animals without any treatment (normal), HFD-fed animals without any treatment (disease-control) and Telmisartan-treated animals (10 mg/kg body weight, once daily from t=6 weeks) were included as references. N.s. = not significant.

Figure 8: Effects of HE-700 treatment on plasma triglycerides in STAM mice (STZ injection and HFD-feeding). HE-700 treatment and HE-900 vehicle control treatment (both 1.5 ml/kg body weight, every other day) were started at t=5 weeks or t=6 weeks and continued until the end of the study (t=9 weeks). Chow-fed animals without any treatment (normal), HFD-fed animals without any treatment (disease-control) and Telmisartan-treated animals (10 mg/kg body weight, once daily from t=6 weeks) were included as references. N.s. = not significant.

Figure 9: Effects of HE-700 treatment on liver triglycerides in STAM mice (STZ injection and HFD-feeding). HE-700 treatment and HE-900 vehicle control treatment (both 1.5 ml/kg body weight, every other day) were started at t=5 weeks or t=6 weeks and continued until the end of the study (t=9 weeks). Chow-fed animals without any treatment (normal), HFD-fed animals without any treatment (disease-control) and Telmisartan-treated animals (10 mg/kg body weight, once daily from t=6 weeks) were included as references. N.s. = not significant.

Figure 10: Effects of HE-700 treatment on the NAFLD activity score (NAS) in STAM mice (STZ injection and HFD-feeding). HE-700 treatment and HE-900 vehicle control treatment (both 1.5 ml/kg body weight, every other day) were started at t=5 weeks or t=6 weeks and continued until the end of the study (t=9 weeks). Chow-fed animals without any treatment (normal), HFD-fed animals without any treatment (disease-control) and Telmisartan-treated animals (10 mg/kg body weight, once daily from t=6 weeks) were included as references.

Figure 11: Effects of HE-700 treatment on the fibrosis area in STAM mice (STZ injection and HFD-feeding). HE-700 treatment and HE-900 vehicle control treatment (both 1.5 ml/kg body weight, every other day) were started at t=5 weeks or t=6 weeks and continued until the end of the study (t=9 weeks). Chow-fed animals without any treatment (normal), HFD-fed animals without any treatment (disease-control) and Telmisartan-treated animals (10 mg/kg body weight, once daily from t=6 weeks) were included as references.

Figure 12: Effects of HE-700 treatment on collagen type 1 in STAM mice (STZ injection and HFD-feeding) displayed as representative microscopic images. HE-700 treatment and HE-900 vehicle control treatment (both 1.5 ml/kg body weight, every other day) were started at t=5 weeks or t=6 weeks and continued until the end of the study (t=9 weeks). Chow-fed animals without any treatment (normal), HFD-fed animals without any treatment (disease-control) and Telmisartan- treated animals (10 mg/kg body weight, once daily from t=6 weeks) were included as references. A selection of collagen type 1 positive signals was highlighted with arrows.

Figure 13: Effects of HE-700 treatment on collagen type 3 in STAM mice (STZ injection and HFD-feeding) displayed as representative microscopic images. HE-700 treatment and HE-900 vehicle control treatment (both 1.5 ml/kg body weight, every other day) were started att=5 weeks or t=6 weeks and continued until the end of the study (t=9 weeks). Chow-fed animals without any treatment (normal), HFD-fed animals without any treatment (disease-control) and Telmisartan- treated animals (10 mg/kg body weight, once daily from t=6 weeks) were included as references. A selection of collagen type 3 positive signals was highlighted with arrows.

Figure 14: Effects of HE-700 treatment on Gr-l-positive cells in STAM mice (STZ injection and HFD-feeding) displayed as representative microscopic images. HE-700 treatment and HE-900 vehicle control treatment (both 1.5 ml/kg body weight, every other day) were started at t=5 weeks or t=6 weeks and continued until the end of the study (t=9 weeks). Chow-fed animals without any treatment (normal), HFD-fed animals without any treatment (disease-control) and Telmisartan- treated animals (10 mg/kg body weight, once daily from t=6 weeks) were included as references. A selection of Gr-1 positive signals was highlighted with arrows.

EXAMPLES

The invention shall now be illustrated by the following Examples which shall, whatsoever, not be construed as limiting the scope of the invention in any way.

Example 1: NASH mouse model (genetic modification + HFD-feeding)

70 male Ldlr-/-.Leiden mice (age 13-16 weeks) were used. Animals were housed in makrolon cages (2-4 mice per cage). The experiment was carried out in animal rooms with relative humidity 50-60%, temperature ~21°C and light cycle 7 am to 7 pm. Mice were supplied with food and tap water ad libitum.

An energy-dense high-fat diet (HFD) containing 20 kcal% protein, 35 kcal% carbohydrate and 45 kcal% fat from lard (all w/w) was used to induce NASH (D12451, Research Diets Inc., New Brunswick, NJ, USA). A standard chow diet containing 33 kcal% protein, 58 kcal% carbohydrate and 9 kcal% fat was used as a healthy reference (R/M-H, Ssniff Spezialdiaten GmbH, Soest, Germany).

The 70 male Ldlr-/-.Leiden mice were matched into the following groups:

1. Chow (n= 10)

2. HFD + vehicle control (HE-900 1.5 ml/kg i.p. 3 times/week) start at t=0 (n=15)

3. HFD + HE-700 (HE-700 1.5 ml/kg i.p. 3 times/week) start at t=0 (n=15)

4. HFD + vehicle control (HE-900 1.5 ml/kg i.p. 3 times/week) start at t=6 (n=15)

5. HFD + HE-700 (HE-700 1.5 ml/kg i.p. 3 times/week) start at t=6 (n=15)

For all experiments described herein, at the beginning of the study (t=0 weeks) mice were matched into four groups (1, 2, 3 and remainder) based on body weight (primary matching parameter), plasma cholesterol and blood glucose. In week 6, the remainder mice were matched into group 4 and 5 based on the same parameters.

Treatment (HE-700, Hepar comp., Biologische Heilmittel Heel GmbH, DE) and vehicle control (HE-900) were administered by intraperitoneal injection 3 times per week (on Monday, Wednesday and Friday, between 10:00 a.m. and 12:00 noon). On each treatment day, new ampoules were opened and used (any liquid remaining after treatment was discarded).

Both HE-700 and HE-900 were administered at a dose of 1.5 ml/kg body weight. The injection volumes of HE-700 and HE-900 were calculated based on the average body weight of each experimental group and were adapted according to body weight measurements in week 0, 2, 4, 6, 8, 12, 16, 20 and 24 of the study (i.e. 2-week intervals at the beginning of the study when body weight increases, and 4-week intervals when body weight is more stable).

Animal welfare was monitored daily. Body weight was measured at week 0, 2, 4, 6, 8, 12, 16, 20 and 24. Food intake (per cage) was monitored at week 0, 4, 8, 12, 16, 20 and 24. Mice were sacrificed (un-fasted) by gradual-fill CO2 asphyxiation after 24 weeks of chow/HFD-feeding.

Mice were sacrificed and analyzed as follows:

Liver isolation

The liver was isolated and total liver weight was determined. The medial lobe was isolated, fixed in formalin and embedded in paraffin for histological evaluation. The left lobe was isolated, split in four parts, snap-frozen in liquid N2 and stored at -80°C. One part was used for RNA isolation and the other 3 parts were stored for potential additional analyses. The rest of the liver was also snap-frozen in liquid N2 and stored at -80°C for potential additional analyses.

White adipose tissue isolation

White adipose tissue depots (epididymal and mesenteric adipose tissues) were isolated and weighed. One part of each depot was fixed in formalin and embedded in paraffin for histological evaluation. The other part of each depot was snap frozen in liquid N2 and stored at -80 °C.

Adipose tissue analyses

Paraffin-embedded cross-sections (5 pm) of the perigonadal and the mesenteric white adipose tissue were stained with hematoxylin-phloxine-saffron and digitised using a slide scanner (Aperio AT2, Leica Biosystems, Amsterdam, the Netherlands). Adipose tissue morphometry (average adipocyte size and adipocyte size distribution) and inflammation (number of crown-like structures; CLS per 1000 adipocytes) were analysed as described previously (Jacobs et al., 2019), using the automated image analysis software Adiposoft for morphometry analyses (Galarraga et al., 2012) and counting the number of CLS in the same fields used for morphometry analyses.

Histopathological analysis of NASH

Formalin-fixed and paraffin-embedded cross-sections (3 pm) were stained with haematoxylin and eosin and scored blindly by a board-certified pathologist using an adapted grading method for human NASH (Kleiner et al., 2005; Liang et al., 2014). Briefly, two cross-sections/mouse were examined and the level of microvesicular steatosis, macrovesicular steatosis and hepatocellular hypertrophy was determined relative to the liver area analyzed (expressed as a percentage). Hepatic inflammation was assessed by counting the number of inflammatory foci per field at a 100* magnification in five non-overlapping fields per specimen, expressed as aggregates per mm ² . Neutrophil staining

Analysis of hepatic neutrophil counts was performed by immunohistochemical staining for Ly 6G/Ly 6C (with FITC-conjugated rat anti-mouse monoclonal antibody, eBioscience/Thermo Fisher; followed by rabbit anti-FITC recombinant monoclonal antibody, Invitrogen/Thermo Fisher; and Bright Vision Poly-HRP goat anti-rabbit antibody, VWR International B.V., Amsterdam, the Netherlands). Neutrophils were quantified by counting the number of Ly-6G/Ly- 6C-positive inflammatory foci per field at lOOx magnification in five non-overlapping fields per specimen, expressed as aggregates per mm ² . Based on the results from the gene expression analyses, this analysis was performed for all mice from group 1 (chow), group 4 (HFD+HE-900 late) and group 5 (HFD+HE-700 late).

Analysis of liver lipids

Intrahepatic levels of triglycerides, cholesteryl esters and free cholesterol were determined in freshly prepared liver homogenates by high-performance thin-layer chromatography (HPTLC). Based on the results from the gene expression analyses, this analysis was performed for all mice from group 1 (chow), group 4 (HFD+HE-900 late) and group 5 (HFD+HE-700 late). Lipids were extracted from liver homogenates using methanol and chloroform (following the Bligh and Dyer method (Bligh & Dyer, 1959). After extraction, lipids were separated by HPTLC on silica gel plates. Lipid spots were stained with color reagent (5 g of MnC124H2O, 32 ml of 95-97% H2SO4 added to 960 ml of CH3OH/H2O) and triglycerides, cholesteryl esters and free cholesterol were quantified using a Chemidoc Touch imaging system and Image Lab software (both Bio Rad Laboratories, Veenendaal, the Netherlands). Liver lipids were expressed as pg lipid per mg liver tissue.

Calculation of the net cholesterol production

Feces were collected per cage over a period of 3-4 days in week 16 and week 24 of the study. Food intake was measured during the same period. In short, feces were lyophilized and ground before further processing. For the neutral sterol analyses, feces were treated with alkaline methanol at 80°C for 2 hours, with 5a-cholestane as an internal standard. Neutral sterols were then extracted with petroleum ether and subsequently silylated by DMF-Sil-Prep (Grace Davison Discovery Sciences, Deerfield, USA). For analysis of bile acids, feces were treated with alkaline methanol at 80°C for 120 minutes, with nor-hyodeoxy cholate as an internal standard. Samples were applied to a Sep-Pak Cl 8 solid-phase extraction cartridge (Waters Corporation, Wexford, Ireland) after which bile acids were eluted with methanol and then desiccated. Next, bile acids were derivatized by incubation with trifluoroacetic anhydride and l,l,l,3,3,3-hexafluoro-2-propanol for 1 hour at 60°C and then desiccated and reconstituted in hexane. Both neutral sterols and bile acids were separated on a 25 m * 0.25 mm capillary GC column (CP-Sil 5CB, Varian Chrompack International, Middelburg, the Netherlands) in a 436-GC gas chromatograph with a CP8400 Autosampler (Scion Instruments NL BV, Goes, the Netherlands). The injector and the flame ionisation detector were kept at 300°C for both analyses and the column temperature was programmed from 240 to 280°C for neutral sterol separation and from 230 to 280°C for bile acid separation. Quantitation was based on the area ratio of each individual neutral sterol or bile acid to the respective internal standard. The net cholesterol production was calculated as follows: cholesterol excretion (neutral sterol + bile acid excretion) - dietary cholesterol intake and expressed as pmol/mouse/day.

Statistics

Statistical analyses were performed using SPSS Statistics 24.0 (IBM, Armonk, NY, USA).

Normal distribution of variables was analyzed with the Shapiro-Wilk test, assuming normality at p>0.05. To test equality of variances, Levene’s test of homogeneity of variances was used, assuming equal variances at p>0.05.

For normally distributed variables with equal variances, differences between groups were analyzed by one-way analysis of variance (ANOVA) followed by Dunnet’s post-hoc tests. For normally distributed variables with unequal variances, differences between groups were analyzed by analysis of variance (Brown-Forsythe) and Dunnett’s T3 post-hoc tests.

The non-parametric Kruskall- Wallis test followed by post-hoc with Mann- Whitney U tests was used to determine differences between groups for variables that were not normally distributed.

The early (starting at t=0 weeks) and late (starting at t=6 weeks) treatment with HE-700 were treated as independent analyses that both include the chow group. All comparisons were made with the respective HE-900 control.

Correlation analyses were performed by Spearman’s rank correlation analysis.

Technical errors were excluded. Statistical outliers were excluded for the analysis of lobular inflammation, the hepatic transcriptome, the hepatic neutrophil aggregates and the liver lipids. Data are represented as means ± SD.

Hepatic gene expression analysis

RNA isolation: RNA was isolated from snap-frozen liver tissue samples (left lobe) from all mice using RNA-Bee Total-RNA Isolation Kit (Bio-Connect, Huissen, the Netherlands). RNA concentration was determined spectrophotometrically using Nanodrop 1000 (Isogen Life Science, De Meern, the Netherlands) and RNA quality was assessed using a 2100 Bioanalyzer (Agilent Technologies, Amstelveen, the Netherlands). Next generation sequencing: Based on the results from the histological analysis of hepatic inflammation, next generation RNA sequencing was performed on all mice from group 1 (chow, n=10), group 4 (HFD+HE-900 late, n=15) and group 5 (HFD+HE-700 late, n=15). For this, RNA was used to generate strand-specific mRNA seq libraries for Next Generation Sequencing according to manufacturer’s protocol by service provider GenomeScan B.V. (Leiden, the Netherlands). Libraries were multiplexed, clustered, and sequenced on a HiSeq4000 system (Illumina, San Diego, CA, USA) with a single-read 75 cycles sequencing protocol, 12 million reads per sample and indexing.

Upstream regulator analysis: The number of differential expressed genes (DEG) between groups was determined using an established statistical analysis procedure (DEseq2 pipeline). These DEG were determined using a statistical cut-off of p<0.01 for the following two comparisons:

• HFD+HE-900 vs chow (effects of HFD-feeding in the Ldlr-/-. Leiden model)

• HFD+HE-700 vs HFD+HE-900 (treatment effect of HE-700)

The upstream regulator analysis tool of IP A was used to assess the activity of transcription factors or other upstream regulators essentially. A negative Z-score <-2 indicates a reduced transcriptional activity based on the direction of gene expression changes of target genes. A positive Z-score >2 indicates activation of the upstream regulator.

Example 2: Effects of HE-700 in a NASH mouse model (genetic modification + HFD-feeding) Adipose tissue hypertrophy

HFD feeding markedly and significantly increased the average adipocyte size of mesenteric white adipose tissue (mW AT; 3532.4 ± 588.0 pm ² in HFD+HE-900 early, 3133.7 ± 713.8 pm ² in HFD+HE-900 late, both p<0.001 vs. chow) in comparison to chow controls (1730.6 ± 312.2 pm ² ). The average adipocyte size of mW AT was not affected when treatment started from t=6 weeks (2954.0 ± 759.4 in HFD+HE-700 late, Figure IB) but was significantly reduced when treatment started from t=0 weeks (2992.8 ± 714.4 pm ² in HFD+HE-700 early, p<0.05 vs. HFD+HE-900 early, Figure 1A).

Adipose tissue inflammation

HFD feeding also markedly and significantly increased the number of crown-like structures (CLS) in the epididymal white adipose tissue (eWAT; 20.43 ± 9.64 CLS/1000 adipocytes in HFD+HE- 900 early, 11.13 ± 7.93 CLS/1000 adipocytes in HFD+HE-900 late, both p<0.001 vs. chow) in comparison to chow controls (1.10 ± 1.01 CLS/1000 adipocytes). Treatment with HE-700 did not affect the number of CLS when treatment started from t=6 weeks (13.44 ± 9.05 CLS/1000 adipocytes in HFD+HE-700 late, Figure 2B), but it was significantly reduced in eWAT when treatment started from t=0 weeks (12.54 ± 9.92 CLS/1000 adipocytes in HFD+HE-700 early, p<0.05 vs. HFD+HE-900 early, Figure 2A).

Lobular inflammation

The number of inflammatory cell clusters was very low in chow controls (0.1 ± 0.1 inflammatory aggregates per mm ² liver tissue). HFD feeding markedly and significantly increased the number of inflammatory cell aggregates (1.8 ± 1.0 inflammatory aggregates per mm ² liver tissue in HFD±HE-900 early, 2.0 ± 1.3 inflammatory aggregates per mm ² liver tissue in HFD±HE-900 late, both p<0.001 vs. chow). While treatment with HE-700 from t=0 weeks did not affect the number of inflammatory cell clusters (2.0 ± 1.6 inflammatory aggregates per mm ² liver tissue; Figure 3 A), the number of inflammatory cell clusters was significantly reduced by about 50% in mice treated with HE-700 from t=6 weeks (0.9 ± 0.4 inflammatory aggregates per mm ² liver tissue; Figure 3B).

Hepatic transcriptomics analysis

To further investigate these potential anti-inflammatory effects of HE-700, next generation sequencing (NGS) of liver RNA was performed followed by an upstream regulator analysis.

Based on the observed reduction in hepatic inflammation specifically in the animals treated with HE-700 from t=6 weeks onwards, Next Generation Sequencing (NGS) of the liver transcriptome was performed on all animals from the chow group, the HFD±HE-900 late group and the HFD±HE-700 late group. The animals that were statistical outliers for this parameter (1 mouse in the HFD±HE-900 late group and 2 mice in the HFD±HE-700 late group) were excluded as biological outliers and were not included in statistical analysis of the sequencing data.

Principal Component Analysis (PCA) was used to illustrate the transcriptome per mouse and to visualize group clustering and/or identify potential technical outliers. This PCA analysis identified 1 technical outlier (mouse 76, in the HFD±HE-900 late group), which was excluded from further statistical analysis. The PCA analysis further showed animals of the chow group cluster together, indicating a similar gene expression profile. This is expected and typically observed for chow- treated groups. The HFD-treated animals displayed gene expression profiles that were distinct from those of chow. The dots that represent the transcriptome profiles of HFD±HE-900 late and HFD±HE-700 late mice did not form discrete clusters (data not shown) indicating that the effects of HE-700 need to be dissected via in-depth analysis of the transcriptome profiles.

To investigate the effects of HE-700 treatment, the number of differential expressed genes (DEG) between groups was determined using an established statistical analysis procedure (DEseq2 pipeline). DEG were determined using a statistical cut-off of p<0.01 for the following two comparisons: • HFD+HE-900 vs chow (effects of HFD-feeding in the Ldlr-/-. Leiden model)

• HFD+HE-700 vs HFD+HE-900 (treatment effect of HE-700)

These data show that HFD feeding (+ HE-900 vehicle injections) results in 4112 differentially expressed genes relative to chow control animals. This constitutes a pronounced HFD effect that is consistent with typical inductions of genes by HFD in Ldlr-/-. Leiden mice. In comparison with HFD+HE-900 late mice, HE-700 treatment significantly affected the expression of 50 genes (Table 1).

Table 1: The 50 DEG (p<0.01) in HFD+HE-700-treated mice. A fold-change > 1 indicates upregulation of a gene, a fold-change < 1 indicates downregulation. For transcripts that are not annotated and are unknown with respect to their function (indicated as #NA), the ensembl gene ID (ENSMUS) has been provided. A -log(p-value) of >2 is considered statistically significant.

Closer investigation of these 50 DEG in the context of NASH pathogenesis, revealed that a number of these genes regulated by HE-700 are involved in lipid processing, e.g. Abcg8 (a transporter for cholesterol excretion into bile (Wang et al., 2015)) and Pla2g4c (a cytosolic phospholipase that hydrolyzes the ester bond of glycerophospholipids to produce free fatty acids and lysophospholipids (Yeagle, 2016)). Other DEG are involved in inflammatory processes, such as Cxcll (a chemokine with neutrophil-attractant properties (Marra & Tacke, 2014)), Socs3 (part of a negative feedback mechanism to limit the pro-inflammatory response (Zhou et al., 2017)) and Gzma (the most abundant serine protease in cytotoxic granules of cytotoxic T cells and natural killer cells (Lieberman, 2011)). For most of the genes regulated by HE-700, the direction of regulation was opposite to the direction of regulation in HFD+HE-900 vs chow (i.e. these genes were counter regulated by HE-700), indicating that HE-700 reversed the disease-inducing effects of HFD feeding. Since these effects were not limited to a specific biological process, these data imply that a pronounced effect on a specific pathway is rather unlikely.

Next it was investigated whether HE-700 may have an effect on the predicted activation state of upstream regulators (e.g. transcription factors, cytokines) based on the gene expression pattern downstream of each upstream regulator. This analysis compares the observed gene expression pattern with the expected gene expression pattern if a specific upstream regulator would be activated or inactivated. The top 25 upstream regulators in HFD+HE-700 vs HFD+HE-900 are shown in Table 2. This analysis revealed that while many upstream regulators were significantly enriched in HE-700-treated animals relative to vehicle controls (i.e. more genes downstream of the regulator were differentially expressed than what would be expected to occur by chance) only one of these upstream regulators showed a consistency in its direction of regulation (i.e. the pattern of downstream genes is consistent with an activation or inactivation of the regulator). This effect was significant for the upstream regulator ‘cholesterol’, which was predicted to be activated in HFD+HE-900 vs chow (-log(p-value) = 20.9; z-score = 5.5) and was predicted to be inactivated in HFD+HE-700 vs HFD+HE-900 (-log(p-value) = 3.5; z-score = -2.0), indicating that the predicted HFD-induced effects downstream of intrahepatic cholesterol (e.g. possibly due to an increase in intrahepatic cholesterol or inappropriate cholesterol handling) may be reversed by treatment with HE-700.

Table 2: Top 25 upstream regulators in HFD+HE-700 vs HFD+HE-900. The -log(p-value) indicates significance of enrichment of genes downstream of the upstream regulator. A -logovalue) of >2 is considered statistically significant. The z- value indicates the direction of regulation, i.e. a positive z- value >2 indicates predicted activation of the upstream regulator and a negative z- value <-2 indicates predicted inactivation of the upstream regulator.

Hepatic neutrophil aggregates

To analyze whether the observed effect of HE-700 on Cxcll (a chemokine with neutrophilattractant properties (Marra & Tacke, 2014)) could be confirmed immunohistochemically, the number of neutrophil aggregates was assessed via GR-1 staining. In line with the RNA sequencing analyses, this analysis was performed on liver samples from all mice from group 1, group 4 (HFD+HE-900 late) and group 5 (HFD+HE-700 late). In comparison to chow fed animals (0.16 ± 0.11 neutrophil aggregates / mm ² liver tissue), HFD feeding led to a significant increase in neutrophil aggregates (2.10 + 1.05 neutrophil aggregates / mm ² liver tissue in HFD+HE-900 late, p<0.001 vs. chow). Treatment with HE-700 from t=6 weeks significantly reduced the number of neutrophil aggregates in the liver (1.16 ± 0.46 neutrophil aggregates / mm ² liver tissue in HFD+HE-700 late, p<0.01 vs. HFD+HE-900 late, Figure 4). The reduction of neutrophil aggregates with HE-700 is in line with the observed effect in the NGS analysis.

Liver lipids analysis

To investigate whether the observed effect of HE-700 on cholesterol as an upstream regulator (on the gene expression level) could be confirmed biochemically, the intrahepatic level of triglycerides (TG), cholesteryl esters (CE) and free (non-esterified) cholesterol (FC) was analyzed using HPTLC. In line with the RNA sequencing analyses, this analysis was performed on liver samples from all mice from group 1 (chow), group 4 (HFD+HE-900 late,) and group 5 (HFD+HE-700 late). This analysis showed that hepatic TG (Figure 5A), CE (Figure 5B) and FC (Figure 5C) levels were low in chow-fed animals (6.8 ± 3.8 pg TG/mg tissue; 1.0 ± 0.4 pg CE/mg tissue; 1.7 ± 0.2 pg FC/mg tissue) and were significantly increased in HFD+HE-900 controls (28.7 ± 5.7 pg TG/mg tissue; 3.8 ± 0.9 pg CE/mg tissue; 2.5 ± 0.3 pg FC/mg tissue; all p<0.001 vs. chow). Treatment with HE-700 did not affect TG levels relative to HE-900 controls (25.9 ± 5.6 pg/mg tissue), tended to reduce hepatic CE (3.2 ± 0.7 pg/mg tissue; p=0.057 vs. HFD+HE-900 late) and significantly reduced levels of FC in the liver (2.2 ± 0.4 pg/mg tissue; p<0.01 vs HFD+HE-900 late) thereby confirming the effect of HE-700 on hepatic cholesterol levels observed in the NGS analysis. Free cholesterol levels were found to correlate significantly with hepatic inflammatory aggregates, thereby providing a potential rationale for the observed anti-inflammatory effects of HE-700 in the liver (Figure 5D)

Analysis of cholesterol balance

To further substantiate the observed effect on intrahepatic cholesterol, the cholesterol balance (net cholesterol production = cholesterol excretion via neutral sterols and bile acids - dietary cholesterol intake) was calculated. In HFD fed Ldlr-/-. Leiden mice more cholesterol was excreted via feces than ingested via diet, indicating that there is a net cholesterol production in the body (2.28 ± 0.68 pmol/mouse/day in HFD+HE-900 t=24 weeks, 1.49 ± 0.49 pmol/mouse/day in HFD+HE-900 t=16 weeks). The net cholesterol production was significantly reduced with HE- 700 at t=16 weeks (0.72 ± 0.27 pmol/mouse/day in HFD+HE-700 t=16 weeks, p<0.01 vs. HFD+HE-900 t=16 weeks, Figure 6 A) and a trend towards reduction was detected with HE-700 at t=24 weeks (1.48 ± 0.62 pmol/mouse/day in HFD+HE-700 t=24 weeks, p=0.06 vs. HFD+HE- 900 t=24 weeks, Figure 6B). These results are in line with the observed effects on intrahepatic cholesterol accumulation.

Summary

HE-700 treatment has anti-inflammatory molecular (e.g. Cxcll expression) and cellular (e.g. lobular inflammation and neutrophil content) effects in obese Ldlr-/-. Leiden mice with NASH. A reduction of lipotoxic lipid species, i.e. free cholesterol, may provide a rationale for the observed NASH-attenuating effect.

Example 3: NASH mouse model (streptozotocin + HFD-feeding)

C57BL/6 mice (15-day-pregnant female) were obtained from Japan SLC (Shizuoka, Japan). The animals were maintained in polymethylpentene cages CL-0133 (CLEA Japan) under controlled conditions of temperature (23 ± 2°C), humidity (45 ± 10%), lighting (12-hour artificial light and dark cycles; light from 8:00 to 20:00) and air exchange. A high pressure (20 ± 4 Pa) was maintained in the experimental room to prevent contamination of the facility.

NASH was induced in 60 male mice by a single subcutaneous injection of 200 pg streptozotocin (STZ, Sigma-Aldrich, USA) solution 2 days after birth and feeding with high fat diet (HFD, 57 kcal% fat, cat#: HFD32, CLEA Japan, Japan) after 4 weeks of age. Ten male littermates, fed with normal diet and without STZ treatment, were used for the normal group.

Vehicle control and Hepar comp. (HE-700) were administered by intraperitoneal route in a volume of 1.5 ml/kg every other day. Telmisartan was administered by oral route in a volume of 10 mg/kg once daily.

The following study groups were compiled:

Group 1: Normal

Ten normal mice were fed with a normal diet ad libitum without any treatment until 9 weeks of age.

Group 2: Disease-control

Ten NASH mice were fed with the HFD ad libitum without any treatment until 9 weeks of age.

Group 3: Telmisartan

Ten NASH mice were orally administered pure water supplemented with Telmisartan at a dose of 10 mg/kg once daily from 6 to 9 weeks of age.

Group 4: Vehicle (6-9w)

Ten NASH mice were intraperitoneally administered vehicle (HE-900) in a volume of 1.5 ml/kg every other day from 6 to 9 weeks of age.

Group 5: HE-700 (6-9w)

Ten NASH mice were intraperitoneally administered HE-700 in a volume of 1.5 ml/kg every other day from 6 to 9 weeks of age.

Group 6: Vehicle (5-9w)

Ten NASH mice were intraperitoneally administered vehicle (HE-900) in a volume of 1.5 ml/kg every other day from 5 to 9 weeks of age.

Group 7: HE-700 (5-9w)

Ten NASH mice were intraperitoneally administered HE-700 in a volume of 1.5 ml/kg every other day from 5 to 9 weeks of age.

The viability, clinical signs and behavior were monitored daily. Body weight was recorded before the treatment. Mice were observed for significant clinical signs of toxicity, moribundity and mortality approximately 60 minutes after each administration. The animals were sacrificed by exsanguination through direct cardiac puncture under ether anesthesia (Wako Pure Chemical Industries).

Mice were analyzed as follows: Measurement of plasma triglyceride level

For plasma biochemistry, blood was collected in polypropylene tubes with anticoagulant (Novo- Heparin, Mochida Pharmaceutical, Japan) and centrifuged at l,000xg for 15 minutes at 4°C. The supernatant was collected and stored at -80°C until use. Plasma triglyceride levels were measured by FUJI DRI-CHEM 7000 (Fujifilm, Japan).

Measurement of liver triglyceride content

Liver total lipid-extracts were obtained by Folch’s method (Folch et al., 1957). Liver samples were homogenized in chloroform-methanol (2: 1, v/v) and incubated overnight at room temperature. After washing with chloroform-methanol-water (8:4:3, v/v/v), the extracts were evaporated to dryness, and dissolved in isopropanol. Liver TG levels were measured by Triglyceride E-test (Wako Pure Chemical Industries, Japan).

Histopathology

For HE staining, sections were cut from paraffin blocks of liver tissue prefixed in Bouin’s solution and stained with Lillie-Mayer’s Hematoxylin (Muto Pure Chemicals, Japan) and eosin solution (Wako Pure Chemical Industries). NAFLD Activity score (NAS) was calculated according to the criteria of Kleiner (Kleiner et al., 2005). To visualize collagen deposition, Bouin’s fixed liver sections were stained using picro-Sirius red solution (Waldeck, Germany). For quantitative analysis of fibrosis area, bright field images of Sirius red-stained sections were captured around the central vein using a digital camera (DFC280; Leica, Germany) at 200-fold magnification, and the positive areas in 5 fi elds/ section were measured using Image J software (National Institute of Health, USA).

For immunohistochemistry, sections were cut from frozen left lateral liver tissues embedded in Tissue-Tek O.C.T. compound and fixed in acetone. Endogenous peroxidase activity was blocked using 0.03% H2O2 for 5 minutes, followed by incubation with Block Ace (Dainippon Sumitomo Pharma, Japan) for 10 minutes. The sections were incubated with a 50-fold dilution of anti-Gr-1 (BD Bioscience Pharmingen, USA), a 2000-fold dilution of anti-collagen Type 1 (LSL, Japan) or a 2000-fold dilution of anti-collagen Type 3 (LSL) for 1 hour at room temperature. After incubation with secondary antibody (HRP-Goat anti-rat antibody, Invitrogen, USA, or HRP-Goat anti-rabbit antibody, Jackson ImmunoRearch Laboratories, USA), enzyme- substrate reactions were performed using 3, 3’-diaminobenzidine/H2O2 solution (Nichirei, Japan). Statistics

Statistical analyses were performed using Bonferroni Multiple Comparison Test on GraphPad Prism 4 (GraphPad Software, USA). P values < 0.05 were considered statistically significant. A trend or tendency was assumed when a one-tailed t-test returned P values < 0.05. One mouse from the Telmisartan group died during the experiment and was therefore excluded from the statistical analysis. Results were expressed as mean ± SD.

Example 4: Effects of EE-700 in a NASH mouse model (streptozotocin + HFD-feeding)

Liver weight

Mean liver weight significantly increased in the Disease-control (1217.9 ± 202.3 mg), Vehicle (6- 9w) (1359.3 ± 280.1 mg) and Vehicle (5-9w) (1280.5 ± 211.3 mg) groups compared with the Normal group (998.3 ± 107.8 mg). The Telmisartan group showed a decreasing tendency of mean liver weight (1021.7 ±143.6 mg) compared with the Disease-control group. The liver weight tended to decrease in the EE-700 (6-9w) group (1196 ± 65.5 mg) compared with the Vehicle (6- 9w) group. There was no significant difference in mean liver weight between the Vehicle (5-9w) group and the EE-700 (5-9w) group (1171.1 ± 107.4 mg) (Figure 7).

Plasma triglyceride

Plasma triglyceride levels significantly increased in the Vehicle (6-9w) (527.8 ± 329 mg/dL) and Vehicle (5-9w) (378 ± 235.9 mg/dL) groups and tended to increase in the Disease-control group (225.8 ± 186.2 mg/dL) compared with the Normal group (75.9 ± 24.7 mg/dL). There was no significant difference in plasma triglyceride levels between the Disease-control group and the Telmisartan group (348.2 ± 369.3 mg/dL). Plasma triglyceride levels tended to decrease in the HE- 700 (6-9w) group (266 ± 122 mg/dL) compared with the Vehicle (6-9w) group. There was no significant difference in plasma triglyceride levels between the Vehicle (5-9w) group and the EE- 700 (5-9w) group (207.6 ± 223.5 mg/dL) (Figure 8).

Liver triglyceride content

Liver triglyceride contents significantly increased in the Disease-control (20.4 ± 8.1 mg/g liver), Vehicle (6-9w) (23.6 ± 6.6 mg/g liver) and Vehicle (5-9w) (30.2 ± 11.8 mg/g liver) groups compared with the Normal group. The liver triglyceride contents tended to decrease in the Telmisartan group (12.7 ± 6.9 mg/g liver) compared with the Disease-control group. There was no significant difference in liver triglyceride contents between the Vehicle (6-9w) group and the HE- 700 (6-9w) group (27.2 ± 11.7 mg/g liver). Liver triglyceride contents tended to decrease in the HE-700 (5-9w) group (20.5 ± 7.6 mg/g liver) compared with the Vehicle (5-9w) group (Figure 9).

Histology - HE staining

Liver sections from the Disease-control, Vehicle (6-9w) and Vehicle (5-9w) groups exhibited severe micro- and macrovesicular fat deposition, hepatocellular ballooning and inflammatory cell infiltration. Consistent with these observations, NAS significantly increased in the Disease-control group (5.1 ± 1) compared with the Normal group (0 ± 0). The Telmisartan group showed marked improvements in fat deposition, hepatocellular ballooning and inflammatory cell infiltration, with significant reduction in NAS (2.6 ± 1.1) compared with the Disease-control group. The HE-700 (6-9w) group showed marked improvements in hepatocellular ballooning and moderate improvements in fat deposition and inflammatory cell infiltration. NAS significantly decreases in the HE-700 (6-9w) group (3.2 ± 1.1) compared with Vehicle (6-9w) group (5 ± 0.8). The HE-700 (5-9w) group showed marked improvements in fat deposition, hepatocellular ballooning and lobular inflammation (3.6 ± 1.2) compared with the Vehicle (5-9w) group (4.8 ± 0.6) with a significant reduction of NAS (Figure 10).

Histology - Sirius red staining

Liver sections from the Disease-control, Vehicle (6-9w) and Vehicle (5-9w) groups showed increased collagen deposition in the pericentral region of liver lobule compared with the Normal group. The percentage of fibrosis area (Sirius red-positive area) significantly increased in the Disease-control (1.25 ± 0.4 %), Vehicle (6-9w) (1.18 ± 0.4 %) and Vehicle (5-9w) (1.32 ± 0.3 %) groups compared with the Normal group (0.29 ± 0,1%). The fibrosis area significantly decreased in the Telmisartan group (0.69 ± 0.2 %) compared with the Disease-control group. The fibrosis area significantly decreased in the HE-700 (6-9w) group (0.83 ± 0.2 %) compared with the Vehicle (6-9w) group. The fibrosis area significantly decreased in the HE-700 (5-9w) group (0.87 ± 0.2%) compared with the Vehicle (5-9w) group (Figure 11).

Histology - Immunostaining for Collagenes Type 1 and 3 and GR-1

Collagen Type 1 immunostaining revealed that collagen deposition in the sinusoids of the liver lobule increased in the Disease-control, Vehicle (6-9w) and Vehicle (5-9w) groups compared with the Normal group. Telmisartan treatment showed a reduction of the collagen deposition compared with the Disease-control group. HE-700 treatment showed a reduction of the collagen deposition compared with the Vehicle group in both cohorts (Figure 12). Collagen Type 3 immunostaining revealed that collagen deposition in the sinusoids of the liver lobule increased in the Disease-control, Vehicle (6-9w) and Vehicle (5-9w) groups compared with the Normal group. Telmisartan treatment showed a reduction of the collagen deposition compared with the Disease-control group. HE-700 treatment showed a reduction of the collagen deposition compared with the Vehicle group in both cohorts (Figure 13).

Liver sections from the Disease-control, Vehicle group (6-9w) and Vehicle (5-9w) groups showed an increased number of Gr-1 -positive cells around central veins in the liver lobule compared with the Normal group. Lower Gr-1 -positive signals were observed in the Telmisartan group compared with the Disease-control group. Lower Gr-1 -positive signals were observed in the HE-700 (6-9w) group compared with the Vehicle (6-9w) group, and HE-700 (5-9w) group compared with the Vehicle (5-9w) group (Figure 14).

Summary HE-700 treatment has anti-NASH (e.g. NAFLD activity score), anti-inflammatory (e.g. lobular inflammation and neutrophil numbers as demonstrated by Gr-1 -positive signals) and anti-fibrotic (e.g. fibrosis area as measured via Sirius red staining and collagen type 1 and 3) effects in STAM mice with NASH.

CITED LITERATURE

1. Lucas C, Lucas G, Lucas N, Krzowska-Firych J, Tomasiewicz K. A systematic review of the present and future of non-alcoholic fatty liver disease. Clin Exp Hepatol. 2018;4(3): 165-174. doi: 10.5114/ceh.2018.78120

2. Benedict M, Zhang X. Non-alcoholic fatty liver disease: An expanded review. World J Hepatol. 2017;9(16):715-732. doi: 10.4254/wjh.v9.il6.715

3. Iqbal U, Perumpail B, Akhtar D, Kim D, Ahmed A. The Epidemiology, Risk Profiling and Diagnostic Challenges of Nonalcoholic Fatty Liver Disease. Medicines. 2019;6(l):41. doi: 10.3390/medicines6010041

4. Younossi ZM. Non-alcoholic fatty liver disease - A global public health perspective. J Hepatol. 2019;70(3):531-544. doi: 10.1016/j jhep.2018.10.033

5. Yu J, Marsh S, Hu J, Feng W, Wu C. The Pathogenesis of Nonalcoholic Fatty Liver Disease: Interplay between Diet, Gut Microbiota, and Genetic Background. Gastroenterol Res Pract. 2016;2016:2862173. doi: 10.1155/2016/2862173

6. Stefan N, Haring H-U, Cusi K. Non-alcoholic fatty liver disease: causes, diagnosis, cardiometabolic consequences, and treatment strategies, lancet Diabetes Endocrinol. 2019;7(4):313-324. doi: 10.1016/S2213-8587(18)30154-2

7. Pydyn N, Mi^kus K, Jura J, Kotlinowski J. New therapeutic strategies in nonalcoholic fatty liver disease: a focus on promising drugs for nonalcoholic steatohepatitis. Pharmacol Rep. 2020;72(l):l-12. doi: 10.1007/s43440-019-00020-l

8. Kleiner DE, Brunt EM, Van Natta M, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005;41(6): 1313-1321. doi: 10.1002/hep.20701

9. Liang W, Menke AL, Driessen A, et al. Establishment of a General NAFLD Scoring System for Rodent Models and Comparison to Human Liver Pathology. Sookoian SC, ed. PLoS One. 2014;9(12):el 15922. doi: 10.1371/journal.pone.0115922

10. Bligh EG, Dyer WJ. A RAPID METHOD OF TOTAL LIPID EXTRACTION AND PURIFICATION. Can J Biochem Physiol. 1959;37(8):911-917. doi: 10.1139/o59-099 11. Wang J, Mitsche MA, Liitjohann D, Cohen JC, Xie X-S, Hobbs HH. Relative roles of ABCG5/ABCG8 in liver and intestine. J Lipid Res. 2015;56(2):319-330. doi:10.1194/jlr.M054544

12. Kamisako T, Tanaka Y, Kishino Y, et al. Role of Nrf2 in the alteration of cholesterol and bile acid metabolism-related gene expression by dietary cholesterol in high fat-fed mice. J Clin Biochem Nutr. 2014;54(2):90-94. doi: 10.3164/jcbn,13-92

13. loannou GN. The Role of Cholesterol in the Pathogenesis of NASH. Trends Endocrinol Metab. 2016;27(2):84-95. doi:10.1016/j.tem.2015.11.008

14. Marra F, Svegliati-Baroni G. Lipotoxicity and the gut-liver axis in NASH pathogenesis. J Hepatol. 2018;68(2):280-295. doi: 10.1016/j.jhep.2017.11.014

15. FOLCH J, LEES M, SLOANE STANLEY GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957;226(l):497-509.

16. Sharma P, Arora A. Clinical presentation of alcoholic liver disease and non-alcoholic fatty liver disease: spectrum and diagnosis. Transl Gastroenterol Hepatol. 2020;5: 19-19. doi: 10.21037/tgh.2019.10.02

17. Yeagle PL. Lipid-Protein Interactions in Membranes. In: The Membranes of Cells. Elsevier; 2016:291-334. doi:10.1016/B978-0-12-800047-2.00012-7

18. Marra F, Tacke F. Roles for Chemokines in Liver Disease. Gastroenterology. 2014;147(3):577-594.el. doi: 10.1053/j.gastro.2014.06.043

19. Zhou D, Chen L, Yang K, Jiang H, Xu W, Luan J. SOCS molecules: the growing players in macrophage polarization and function. Oncotarget. 2017;8(36). doi : 10.18632/oncotarget.19940

20. Lieberman J. Granzyme A activates another way to die. Immunol Rev. 2010;235(l):93-104. doi: 10.1111/j.0105-2896.2010.00902.

20. Jacobs SAH, Gart E, Vreeken D, et al. Sex-Specific Differences in Fat Storage, Development of Non-Alcoholic Fatty Liver Disease and Brain Structure in Juvenile HFD-Induced Obese Ldlr- /-. Leiden Mice. Nutrients. 2019;l 1(8). doi: 10.3390/nul l081861

21. Galarraga M, Campion J, Munoz-Barrutia A, et al. Adiposoft: automated software for the analysis of white adipose tissue cellularity in histological sections. J Lipid Res. 2012;53(12):2791- 2796. doi: 10.1194/jlr.D023788 22. Amedeo Lonardo, Alessandro Mantovani, Simonetta Lugari, and Giovanni Targher: NAFLD in Some Common Endocrine Diseases: Prevalence, Pathophysiology, and Principles of Diagnosis and Management. Int J Mol Sci. 2019 Jun; 20(11): 2841