Substances for the treatment of fatty liver-related conditions

Abstract

Compositions for the treatment of fatty liver disease and related disorders are provided. [Solution] A composition comprising: A) serine; B) N-acetylcysteine; C) carnitine; and D) nicotinamide riboside or nicotinamide, wherein the molar ratio of A) to D) is 250:1 to 1.5:1 and the molar ratio of A) to B) is 16:1 to 1:4, and wherein the substances included in groups A) to D) are at least 25% of the dry weight of the composition.

Description

[Technical Field]

[0001] The present invention relates to the treatment of fatty liver disease and related disorders. [Background technology]

[0002] Hepatic steatosis (HS) is defined as the accumulation of fat in the liver without evidence of hepatocellular injury, and it is the most common chronic liver disease worldwide (Vetelainen et al., 2007). HS is a hallmark of nonalcoholic fatty liver disease (NAFLD), which is strongly associated with obesity, insulin resistance, type 2 diabetes (T2D), and cardiovascular disease (Ratziu et al., 2010). Up to 30% of NAFLD patients develop nonalcoholic steatohepatitis (NASH), a serious condition in which inflammation and scarring can ultimately lead to cirrhosis and hepatocellular carcinoma (HCC) (Dyson et al., 2014).

[0003] The underlying molecular mechanisms leading to the development of HS and its progression to severe liver damage remain elusive, which limits the identification of drug targets and the discovery of biomarkers that can be used to design effective therapeutic strategies. Summary of the Invention [Problem to be solved by the invention]

[0004] Currently, there are few pharmaceutical treatments for HS and its associated clinical conditions (Machado & Cortez-Pinto, 2012), and we recognized that an integrated systems biology-based approach could help address these important unmet medical needs. In this context, genome-scale metabolic models (GEMs) can be used to gain more insight into the molecular mechanisms involved in the development of HS and related disorders, which in turn can enable therapeutic discovery. GEMs are collections of biochemical reactions known to occur in specific cells / tissues, and these models have been used to integrate cellular, physiological, and clinical data to reveal the molecular mechanisms underlying metabolic disorders.

[0005] We designed a therapeutic strategy for NAFLD based on our understanding of the pathophysiology of dyslipidemia. GEM iHepatocytes2322 contain extensive information on lipid metabolism (Mardinoglu et al., 2014). This is necessary to investigate the impact of excessive amounts of lipids on the molecular mechanisms underlying NAFLD. Therefore, this GEM can be used as a platform to study lipoprotein dynamics and their potential impact on hepatic metabolism.

[0006] To clarify the underlying metabolic disorders in NAFLD, we investigated metabolic differences in the liver between subjects with different degrees of HS by studying the dynamics of lipid metabolism, taking into account the interactions between the liver, adipose tissue, muscle, and other peripheral tissues, as well as red blood cells. Using personalized genome-scale metabolic modeling, we elucidated the underlying molecular mechanisms of NAFLD and used them to develop therapeutic strategies.

[0007] Subjects with varying degrees of HS were characterized and VLDL kinetics measured. Subsequently, the VLDL kinetic data were integrated with additional experimentally obtained flux data to simulate liver metabolism for each subject using liver GEM. Correlations between the predicted intracellular fluxes of hepatic and HS were then evaluated to detect metabolic abnormalities in NAFLD. Systems-level analysis indicated that alterations in NAD+ and GSH metabolism (increased need for NAD and GSH) are a common feature of NAFLD. Therefore, it was hypothesized that subjects with NAFLD have reduced de novo GSH synthesis, possibly due to limited glycine availability in the fasting state. Plasma metabolomics analysis showed that plasma levels of glycine, as well as serine, betaine, and N-acetylglycine (which can be converted to glycine), were lower in subjects with high HS compared to subjects with low HS. Furthermore, analysis of the metabolomics data revealed a significant negative correlation between plasma levels of glycine, serine, betaine, and N-acetylglycine and HS. In mouse studies, supplementation with precursors of NAD+ and GSH was shown to significantly reduce HS. Finally, in a proof-of-concept human study, HS was significantly reduced, while markers of liver function were significantly improved, in patients with NAFLD after supplementation with serine (a precursor to glycine).

[0008] Serine derived from the glycolytic branch can be converted to glycine, which then uses THF to provide the carbon unit for one-carbon metabolism. It has previously been shown that NAFLD patients and controls have similar folate levels, and therefore we conclude that THF is unlikely to be limiting for glycine biosynthesis.

[0009] Increased release of free fatty acids (FAs) during fasting is a known hallmark of obesity and related disorders such as NAFLD (Karpe et al., 2011; Nestel & Whyte, 1968). We demonstrated that low VLDL excretion, combined with high FA influx into the liver (i.e., high net fat influx (NFI)), significantly impacts flux. Increased GSH turnover, as well as increased fat oxidation, increased oxidative phosphorylation with subsequent increased oxygen demand, and increased ketogenesis, were strongly correlated with high NFI.

[0010] Thus, increases in GSH, NAD+, oxidative phosphorylation, oxygen consumption, and ketone production are all model-predicted demands that would ideally be met to address high HS. If any of these demands cannot be easily met in vivo due to reduced substrate concentrations, cellular health may be compromised. For example, if the predicted demand for GSH at high HS is not met by an increased supply of GSH, redox balance may be at risk of being insufficient for normal cellular health at high HS. Indeed, we have shown that the expression of enzymes involved in GSH formation is significantly lower in obese subjects. Given that our simulations demonstrated an ideal hepatic response to increased HS—upregulation of fat oxidation and increased availability of GSH and NAD+—this offers a therapeutic strategy for NAFLD subjects.

[0011] In this analysis, subjects at highest risk for possible metabolic stress were those with high FA influx and HS. Importantly, HS alone is not a single characteristic that explains high GSH demand, meaning that individuals with high HS are not necessarily at risk. Because metabolic distress was predicted to correlate well only with high NFI and FA influx, it can be argued that subjects with high HS but low FA influx are not necessarily at risk for disease. In fact, the expansion of lipid droplets in the liver is one way to process excess FAs. Thus, the HS process itself could theoretically help reduce metabolic stress in the liver. Similarly, increased VLDL secretion, increased ketone secretion, and increased oxidative phosphorylation are all ways the liver can process excess FAs.

[0012] Through systems-level analysis in mice, it has been observed that glycine is the limiting substrate for the de novo synthesis of GSH (Mardinoglu et al., 2015). A recent study comparing germ-free and conventionally raised mice showed that the gut microbiota alters the distribution of AA along the gastrointestinal tract and influences the bioavailability of free AA to the host (Mardinoglu et al., 2015b). It has also been shown that microbially induced imbalances in the utilization of AA, particularly serine and glycine, can affect host biological functions. Furthermore, the presence of gut microbiota resulted in increased expression of Nnt in liver, adipose, and gastrointestinal tissues, as well as parallel decreases in plasma and liver glycine levels.

[0013] The data disclosed herein indicate that increased FA release from adipose tissue and decreased VLDL secretion from the liver increase metabolic stress on the liver. Therefore, it is clinically valuable to consider FA release from adipose tissue along with the degree of HS in subjects with HS.

[0014] In conclusion, personalized genome-scale metabolic modeling has been used to elucidate the molecular mechanisms involved in the progression of NAFLD, and predictions have been validated by generating additional plasma metabolomic data. Furthermore, proof-of-concept studies in mice and humans have demonstrated that supplementation with precursors of NAD+ and GSH is useful for the prevention and treatment of HS. [Means for solving the problem]

[0015] Considering the above modeling results, the inventors provided the following insightful therapeutic strategy.

[0016] To eliminate fats, such as those stored in the liver, hepatocytes burn fatty acids through mitochondrial beta-oxidation. L-carnitine can be supplemented to promote the transport of fatty acids into mitochondria. Furthermore, nicotinamide riboside (NR) can be supplemented to promote mitochondrial beta-oxidation, which generates toxic by-products. Hepatocytes naturally produce antioxidants that neutralize harmful by-products. Antioxidant formation is limited by the availability of glycine. Therefore, supplementation with glycine and / or serine (glycine precursors) can increase antioxidant formation. After sufficient supplementation of glycine and / or serine, cysteine becomes limiting for antioxidant formation. Therefore, to further increase antioxidant formation, cysteine and / or N-acetylcysteine (NAC) can be supplemented in addition to glycine and / or serine. Supplementation not only enhances the neutralization of toxic by-products but also promotes fatty acid beta-oxidation.

[0017] Taking into account the metabolic pathways, the inventors have identified the following alternatives to the above substances:

[0018] [Table 1]

[0019] It is not necessary to include all four substances to achieve a therapeutic effect. However, the inventors have identified serine (or one or more of its substitutes) as the most important substance, and NR (or one or more of its substitutes) as the second most important substance. Furthermore, the inventors have found that the optimal daily molar dose is higher for serine than for NR.

[0020] Nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2D) are common conditions that regularly coexist and may synergistically cause adverse outcomes. The presence of both NAFLD and T2D not only increases the likelihood of developing diabetic complications, but also increases the risk of more severe NAFLD, including liver cirrhosis, hepatocellular carcinoma, and death.

[0021] Fatty liver (hepatic steatosis) is the earliest abnormality in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and alcoholic fatty liver disease (AFLD), due either to metabolic syndrome or chronic alcohol abuse in the absence of metabolic risk factors associated with insulin resistance and / or alcohol consumption. If unchecked, both NAFLD and AFLD can lead to steatohepatitis, fibrosis, cirrhosis, hepatocellular carcinoma (HCC), and eventual death.

[0022] Primary hepatic steatosis in NAFLD is associated with metabolic risk factors reflecting metabolic syndrome (MS), such as obesity, insulin resistance and / or dyslipidemia in the majority of patients.

[0023] Therefore, the above-mentioned therapeutic strategies can be used not only for NAFLD and HS, but also for AFLD, type 2 diabetes, obesity, insulin resistance and dyslipidemia.

[0024] Accordingly, the following itemized list of embodiments of the present disclosure is provided.

[0025] 1. A composition comprising: A) serine, glycine, betaine, N-acetylglycine, N-acetylserine, dimethylglycine, sarcosine and / or phosphoserine; B) optionally N-acetylcysteine, cysteine and / or cystine; C) optionally carnitine, deoxycarnitine, γ-butyrobetaine, 4-trimethylammoniobutanal, 3-hydroxy-N6,N6,N6-trimethyl-L-lysine, N6,N6,N6-trimethyl-L-lysine and / or lysine; and D) nicotinamide riboside, quinolinate, deamino-NAD+, nicotinate D-ribonucleotide, nicotinamide D-ribonucleotide, nicotinate D-ribonucleoside, nicotinamide and / or nicotinate, wherein The molar ratio of A) to D) is 250:1 to 1.5:1.

[0026] 2. The composition according to item 1, wherein the molar ratio of A) to B) is from 16:1 to 1:4, for example from 12:1 to 1.5:1, preferably from 10:1 to 3:1.

[0027] 3. The composition according to item 1 or 2, wherein the molar ratio of A) to C) is from 150:1 to 1:1, for example from 100:1 to 4:1, preferably from 50:1 to 8:1, more preferably from 30:1 to 13:1.

[0028] 4. The composition according to any one of items 1 to 4, wherein the molar ratio of A) to D) is 150:1 to 3:1, preferably 90:1 to 10:1, more preferably 50:1 to 20.

[0029] 5. The composition according to any one of items 1 to 4, wherein A) is serine, preferably L-serine.

[0030] 6. The composition of any one of the preceding items, wherein B) is N-acetylcysteine.

[0031] 7. The composition of any one of the preceding items, wherein C) is carnitine.

[0032] 8. The composition of any one of the preceding items, wherein D) is nicotinamide riboside.

[0033] 9. The composition according to any one of the preceding items, which is an aqueous solution or suspension.

[0034] 10. An aqueous solution or suspension comprising: A) serine; B) N-acetylcysteine; C) carnitine; and D) nicotinamide riboside, where the molar ratio of A) to B) is 12:1 to 1:1.5, preferably 10:1 to 3:1; the molar ratio of A) to C) is 100:1 to 4:1, preferably 50:1 to 8:1, more preferably 30:1 to 13:1; The molar ratio of A) to D) is 150:1 to 3:1, preferably 90:1 to 10:1, and more preferably 50:1 to 20:1.

[0035] 11. An aqueous solution or suspension comprising: A) serine; B) optionally N-acetylcysteine and / or cysteine; C) optionally carnitine; and D) nicotinamide riboside, where The molar ratio of A) to D) is 90:1 to 10:1, preferably 50:1 to 20:1, more preferably 45:1 to 25:1.

[0036] 12. The solution or suspension according to any one of items 9 to 11, wherein the concentration of A) is 0.20 to 2.4 mmol / ml, preferably 0.40 to 2.4 mmol / ml, more preferably 0.60 to 2.4 mmol / ml.

[0037] 13. The solution or suspension according to any one of items 9 to 12, wherein the concentration of D) is 0.006 to 0.12 mmol / ml, preferably 0.012 to 0.08 mmol / ml, more preferably 0.018 to 0.07 mmol / ml.

[0038] 14. The solution or suspension according to any one of items 9 to 13, wherein the concentration of B) is 0.09 to 0.90 mmol / ml, for example 0.09 to 0.54 mmol / ml, preferably 0.11 to 0.40 mmol / ml, more preferably 0.013 to 0.30 mmol / ml.

[0039] 15. The solution or suspension according to any one of items 9 to 14, wherein the concentration of C) is 0.009 to 0.38 mmol / ml, for example 0.009 to 0.19 mmol / ml, preferably 0.016 to 0.16 mmol / ml, more preferably 0.028 to 0.38 mmol / ml.

[0040] 16. A package such as a bottle containing the solution or suspension according to any one of items 9 to 15.

[0041] 17. The package according to item 16, wherein the volume of the package is 25 to 1000 ml, for example 50 to 500 ml.

[0042] 18. A composition, solution or suspension according to any one of the preceding items for use in a therapeutic method for the treatment of a subject.

[0043] 19. The composition, solution, or suspension according to item 18, wherein the method of treatment is a method of treating a condition selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease (AFLD), type 2 diabetes, obesity, insulin resistance, and dyslipidemia.

[0044] 20. The composition, solution or suspension according to item 18 or 19, wherein the method of treatment comprises oral administration of the substance.

[0045] 21. The method of treatment, A) at a dose of 0.48 to 24 mmol / kg / day, for example 0.48 to 4.8 mmol / kg / day, for example 1.8 to 4.8 mmol / kg / day, for example 2.9 to 4.6 mmol / kg / day; optionally B) at a dose of 0.31 to 3.05 mmol / kg / day, e.g., 0.31 to 1.84 mmol / kg / day, e.g., 0.43 to 1.23 mmol / kg / day; optionally C) at a dose of 0.031 to 1.24 mmol / kg / day, for example 0.031 to 0.620 mmol / kg / day, for example 0.062 to 0.50 mmol / kg / day, for example 0.093 to 0.37 mmol / kg / day; and D) The composition, solution or suspension according to item 20, comprising oral administration at a dose of 0.020 to 0.39 mmol / kg / day, for example 0.039 to 0.31 mmol / kg / day, for example 0.059 to 0.24 mmol / kg / day.

[0046] 22. A method for treating a condition selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, type 2 diabetes, or obesity, comprising administering to a subject in need thereof: A) serine, glycine, betaine, N-acetylglycine, N-acetylserine, dimethylglycine, sarcosine and / or phosphoserine at a dose of 0.48 to 24.8 mmol / kg / day, such as 0.48 to 4.8 mmol / kg / day, for example 1.8 to 4.8 mmol / kg / day, for example 2.9 to 4.6 mmol / kg / day; B) optionally N-acetylcysteine, cysteine and / or cystine, at a dose of 0.31 to 3.05 mmol / kg / day, for example 0.31 to 1.84 mmol / kg / day, for example 0.43 to 1.23 mmol / kg / day; C) optionally carnitine, deoxycarnitine, gamma-butyrobetaine, 4-trimethylammoniobutanal, 3-hydroxy-N6,N6,N6-trimethyl-L-lysine, N6,N6,N6-trimethyl-L-lysine and / or lysine, at a dose of 0.031 to 0.24 mmol / kg / day, such as 0.031 to 0.620 mmol / kg / day, for example 0.062 to 0.50 mmol / kg / day, for example 0.093 to 0.37 mmol / kg / day; D) The method as described above, comprising oral administration of nicotinamide riboside, quinolinate, deamino-NAD+, nicotinate D-ribonucleotide, nicotinamide D-ribonucleotide, nicotinate D-ribonucleoside, nicotinamide and / or nicotinate at a dose of 0.020 to 0.39 mmol / kg / day, for example, 0.039 to 0.31 mmol, for example, 0.059 to 0.24 mmol / kg / day.

[0047] 23. The method of item 22, wherein the condition is nonalcoholic steatohepatitis (NASH).

[0048] 24. The method according to item 22 or 23, wherein the treatment is carried out for a period of 1 to 12 weeks, for example 2 to 8 weeks.

[0049] 25. A) serine, glycine, betaine, N-acetylglycine, N-acetylserine, dimethylglycine, sarcosine and / or phosphoserine; B) optionally N-acetylcysteine, cysteine and / or cystine; C) optionally carnitine, deoxycarnitine, γ-butyrobetaine, 4-trimethylammoniobutanal, 3-hydroxy-N6,N6,N6-trimethyl-L-lysine, N6,N6,N6-trimethyl-L-lysine and / or lysine, and D) Nicotinamide riboside, quinolinate, deamino-NAD+, nicotinate D-ribonucleotide, nicotinamide D-ribonucleotide, nicotinate D-ribonucleoside, nicotinamide and / or nicotinate 10. The method of claim 1, wherein the compound is a medicament for simultaneous, separate or sequential use in a therapeutic method for the treatment of a subject.

[0050] 26. The substance according to item 25, wherein A) is serine, B) is N-acetylcysteine, C) is carnitine, and D) is nicotinamide riboside.

[0051] 27. The substance according to item 26, wherein the method of treatment is a method of treating a condition selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, type 2 diabetes, obesity, insulin resistance, and dyslipidemia.

[0052] 28. The substance according to any one of items 25 to 27, wherein the method of treatment comprises oral administration of the substance.

[0053] 29. The substance according to item 28, wherein the treatment method comprises oral administration of: A) a dose of 0.48 to 24 mmol / kg / day, for example 0.48 to 4.8 mmol / kg / day, for example 1.8 to 4.8 mmol / kg / day, for example 2.9 to 4.6 mmol / kg / day; optionally B) a dose of 0.31 to 3.05 mmol / kg / day, e.g., 0.31 to 1.84 mmol / kg / day, e.g., 0.43 to 1.23 mmol / kg / day; optionally C) a dose of 0.031 to 1.24 mmol / kg / day, for example 0.031 to 0.620 mmol / kg / day, for example 0.062 to 0.50 mmol / kg / day, for example 0.093 to 0.37 mmol / kg / day; and D) A dose of 0.020-0.39 mmol / kg / day, e.g., 0.039-0.31 mmol / kg / day, e.g., 0.059-0.24 mmol / kg / day. [Brief explanation of the drawings]

[0054] [Figure 1] A) Body mass index (BMI), insulin resistance (HOMA-IR), plasma triglycerides (TG), and alanine aminotransferase (ALT) levels are significantly correlated with independently measured liver fat. B) Subjects are classified into two groups: high HS and low HS. Body mass index (BMI), fasting plasma insulin (FPI), plasma triglycerides (TG), and plasma (ALT) levels are found to be significantly different between the two groups. Data are presented as mean ± SD.

[0055] [Figure 2] To assess the correlation between predicted hepatic intracellular flux and hepatic steatosis (HS) and compare it with A) apolipoprotein B (apoB) and B) triglyceride (TG) content in total VLDL production.

[0056] [Figure 3] Identification of significantly altered metabolites in subjects with high HS. Plasma levels of ~520 metabolites were detected by untargeted metabolomic profiling, and significantly (P value < 0.5) altered metabolites are presented using a volcano plot.

[0057] [Figure 4] The mRNA expression of nicotinamide nucleotide transhydrogenase (NNT), glutathione reductase (GSR), glutamate-cysteine ligase, catalytic subunit (GCLC), and glutamate-cysteine ligase, modifying subunit (GCLM) was measured in livers obtained from 12 morbidly obese subjects undergoing bariatric surgery and 7 healthy individuals.

[0058] [Figure 5]Supplementation with NAD+ and GSH precursors prevents NAFLD. Ten mice were treated for 14 days with NR (400 mg / kg / day), serine (300 mg / kg / day) by oral gavage, and 1 g / L NAC (N-acetyl-L-cysteine) in drinking water. Figure 5a: Liver lipids, including A) triglycerides, B) cholesterol esters, C) ceramides, D) sphingomyelin, and E) phosphatidylethanolamine (normalized to phosphatidylcholine), are shown for mice fed a Western diet (n = 10) and mice supplemented with the cocktail (n = 10). F) Quantification of serum amino acids from the livers of the same mice before and after supplementation. Figure 5b: G) Analysis of molecular species of triglycerides extracted from mouse livers. Results from the control (untreated) group are expressed as 100%, and results from the treatment groups are expressed as % of the control group. Figure 5c: Human plasma H) alanine aminotransferase (ALT), I) aspartate aminotransferase (AST), J) alkaline phosphatase (ALP), and K) triglyceride (TG) levels are shown for each human subject involved in the study before and after serine supplementation. Each study subject received a single oral dose of L-serine (200 mg / kg) per day for 14 days.

[0059] [Figure 6] FIG. 6 shows a model of the biochemical pathways involved in the β-oxidation of fatty acids in hepatocytes, highlighting the effects of serine, NAC, NR, and L-carnitine supplementation. DETAILED DESCRIPTION OF THE INVENTION

[0060] Through individualized modeling of subjects with HS, we observed that the liver has the ability to remove accumulated fatty acids by oxidizing them within the liver. A three-step strategy was developed: i) increasing fatty acid uptake into mitochondria, ii) increasing fatty acid oxidation in mitochondria, and iii) increasing GSH availability (Figure 6). A cocktail or combination of molecular products can be supplemented to boost two or more of these metabolic processes and ultimately reduce the amount of fatty acids in the liver. L-carnitine and NR can be included in the cocktail or combination to increase the movement of fatty acids from the cytosol to mitochondria and to increase the levels of NAD+, which is required for fatty acid oxidation in mitochondria, respectively. Reduced electron transport chain function combined with increased fatty acid oxidation rates may lead to the accumulation of products of incomplete fatty acid oxidation, combined with increased levels of reactive oxygen species, which may contribute to insulin resistance. To avoid this, the inclusion of serine and NAC in the cocktail or combination can increase GSH levels.

[0061] As a first aspect of the present disclosure, there is provided a composition comprising: A) serine, glycine, betaine, N-acetylglycine, N-acetylserine, dimethylglycine, sarcosine and / or phosphoserine; B) optionally N-acetylcysteine, cysteine and / or cystine; C) optionally carnitine, deoxycarnitine, γ-butyrobetaine, 4-trimethylammoniobutanal, 3-hydroxy-N6,N6,N6-trimethyl-L-lysine, N6,N6,N6-trimethyl-L-lysine and / or lysine; and D) Nicotinamide riboside, quinolinate, deamino-NAD+, nicotinate D-ribonucleotide, nicotinamide D-ribonucleotide, nicotinate D-ribonucleoside, nicotinamide and / or nicotinate.

[0062] In one embodiment of the first aspect, the composition comprises A), B), C) and optionally D).

[0063] In group A), serine and glycine are preferred. The most preferred substance in group A) is serine, which is usually provided as L-serine. As shown in the experimental section below, the effects of serine predicted by the model have been confirmed in human and animal studies.

[0064] In group B), N-acetylcysteine (NAC) and cysteine are preferred. The most preferred substance in group B) is NAC. As shown in the experimental section below, the effects of NAC predicted by the model have been confirmed in animal studies.

[0065] The substance of group C) is preferably carnitine, optionally in the form of carnitine salt, for example, carnitine tartrate.Most preferably, the substance of group C) is L-carnitine, optionally in the form of L-carnitine salt, for example, L-carnitine tartrate.The inventors' model shows that subjects with high HS need to increase fatty acid intake (data not shown).Carnitine can be supplemented to achieve this increase in intake.

[0066] The substance of group D) is preferably nicotinamide riboside (NR). As shown in the experimental section below, the effects of NR predicted by the model have been confirmed in animal experiments.

[0067] Preferably, the group A) agents are present in a higher molar amount than the group D) agents. When efficacy and toxicity are also considered (see dosage discussion below), the molar ratio of A) to D) is usually between 250:1 and 1.5:1, typically between 150:1 and 3:1. Preferably, the molar ratio is between 90:1 and 10:1, more preferably between 50:1 and 20:1.

[0068] In embodiments involving group B) substances, the molar ratio of A) to B) is typically 16:1 to 1:4, preferably 12:1 to 1.5:1 or more, and more preferably between 10:1 and 3:1, taking into consideration efficacy and toxicity.

[0069] In embodiments involving substances of group C), taking into consideration efficacy and toxicity, the molar ratio of A) to C) is usually 150:1 to 1:1, typically 100:1 to 4:1, preferably 50:1 to 8:1, more preferably 30:1 to 13:1.

[0070] The above ratios entail that a patient consuming the composition can obtain an appropriate dose of each substance.

[0071] In one embodiment, the composition of the first aspect is a solid, such as a solid powder. Such a powder can be mixed with water, for example, by the patient / consumer, a nurse, or a doctor. However, the composition of the first aspect is conveniently an aqueous solution or suspension ("cocktail"), which facilitates convenient oral administration. Preferably, such an aqueous solution or suspension is ready to drink.

[0072] In a particularly preferred embodiment of the first aspect, there is provided an aqueous solution or suspension comprising: A) serine; B) N-acetylcysteine; C) carnitine; and D) nicotinamide riboside, wherein the molar ratio of A) to B) is 12:1 to 1:1.5, preferably 10:1 to 3:1; the molar ratio of A) to C) is 100:1 to 4:1, preferably 50:1 to 8:1, more preferably 30:1 to 13:1; The molar ratio of A) to D) is 150:1 to 3:1, preferably 90:1 to 10:1, and more preferably 50:1 to 20:1.

[0073] In another particularly preferred embodiment of the first aspect, there is provided an aqueous solution or suspension comprising: A) serine; B) optionally N-acetylcysteine and / or cysteine; C) optionally carnitine; and D) nicotinamide riboside, Here, the molar ratio of A) to D) is 90:1 to 10:1, preferably 50:1 to 20:1, and more preferably 45:1 to 25:1.

[0074] In an embodiment of the solution or suspension according to the first aspect, - the concentration of A) is typically 0.20 to 2.4 mmol / ml, preferably 0.40 to 2.4 mmol / ml, more preferably 0.60 to 2.4 mmol / ml; and / or The concentration of -D) is typically 0.006 to 0.12 mmol / ml, preferably 0.012 to 0.08 mmol / ml, and more preferably 0.018 to 0.07 mmol / ml.

[0075] When contained in the solution or suspension according to the first aspect: -B) the concentration is usually 0.09 to 0.90 mmol / ml, typically 0.09 to 0.54 mmol / ml, preferably 0.11 to 0.40 mmol / ml, more preferably 0.013 to 0.30 mmol / ml; and / or The concentration of -C) is usually 0.009 to 0.38 mmol / ml, typically 0.009 to 0.19 mmol / ml, preferably 0.016 to 0.16 mmol / ml, and more preferably 0.028 to 0.12 mmol / ml.

[0076] The solution or suspension of the first aspect may be provided in packaging for convenient handling and distribution. Furthermore, the volume of such packaging may be such that drinking the entire contents of the package at one time or over the course of a day results in oral administration of an appropriate dose of the substance in the solution or suspension. In one embodiment, the volume of the package is 25 to 1000 ml. A volume of 50 to 500 ml is preferred. If it is intended that the consumer / patient must drink more than one package per day, the volume will typically be relatively small, e.g., 25 to 500 ml, preferably 25 to 400 ml.

[0077] In one embodiment, the packaged solution or suspension contains 48-478 millimoles of A), thereby providing an effective but non-toxic dose of A. In a preferred embodiment, A) is serine in an amount of 5-50 g, more preferably 10-50 g.

[0078] In an alternative complementary embodiment, the packaged solution or suspension contains 2.0 to 39.2 mmol of D) when D) is NR, and 2.0 to 196 mmol of D) when D) is not NR, thereby providing an effective but non-toxic dose of D). In a preferred embodiment, D) is NR in an amount of 0.5 to 10 g, more preferably 1.5 to 6 g.

[0079] When the composition of the first embodiment is a powder, it may be packaged. From the above discussion, it can be seen that such a pack of powder may contain 48 to 478 mmol of A) and / or 2.0 to 39.2 mmol of D) when D) is NR, and 2.0 to 196 mmol of D) when D) is not NR. Furthermore, such a fill powder preferably contains serine in an amount of 5 to 50 g and / or NR in an amount of 0.5 to 10 g. More preferably, such a fill powder contains serine in an amount of 10 to 50 g and / or NR in an amount of 1.5 to 6.0 g.

[0080] The substances of the present disclosure preferably constitute the majority of the composition, solution, or suspension of the first aspect. For example, the substances included in groups A) to D) may amount to at least 10%, such as at least 25%, such as at least 50% by dry weight of the composition, solution, or suspension of the first aspect. In one embodiment, the weight of serine is at least 10%, such as at least 25%, such as at least 40% by dry weight of the composition, solution, or suspension of the first aspect.

[0081] The composition of the first embodiment can include one or more taste-providing agents, such as one or more sweeteners (e.g., sucralose) and / or one or more flavoring agents. It can also include a lubricant, such as a polyethylene glycol lubricant (e.g., Polyglykol 8000 PF (Clariant)).

[0082] From the above description, it can be seen that the composition can be used for therapeutic purposes. In a second aspect of the present disclosure, there is provided a composition, solution or suspension according to the first aspect for use in a therapeutic method for the treatment of a subject.

[0083] The method of treatment can be a method of treating a condition selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease (AFLD), type 2 diabetes, obesity, insulin resistance, and dyslipidemia.

[0084] In a preferred embodiment, the method of treatment is a method of treating a condition selected from the group consisting of NAFLD and AFLD. In a particularly preferred embodiment, the method of treatment is a method of treating non-alcoholic steatohepatitis (NASH), which is part of the group of conditions called NAFLD. NASH is usually considered the most extreme form of NAFLD and is often considered the main cause of cirrhosis.

[0085] In an embodiment of the second aspect, the method of treatment comprises oral administration of said composition, solution or suspension.

[0086] To achieve a therapeutic effect without reaching toxic levels in the human body, the inventors have found the following doses for the substances of the present disclosure: A) Represented by Serin Daily dose range: 50-2000 mg / kg (0.478-24 mmol / kg), preferably not exceeding 500 mg / kg (4.78 mmol / kg) in a single dose Recommended dose: 400 mg / kg / day (3.8 mmol / kg / day) B) Represented by N-acetylcysteine (NAC) Daily dose range: 50-500 mg / kg (0.306-3.06 mmol / kg) Recommended dose: 100 mg / kg / day (0.613 mmol / kg / day) C) Represented by L-carnitine Daily dose range: 5 to 200 mg / kg (0.031 to 1.24 mmol / kg) Recommended dose: 30 mg / kg / day (0.186 mmol / kg / day) D) Represented by nicotinamide riboside (NR) Daily dose range: 5-100 mg / kg (0.0196-0.392 mmol / kg) * Recommended dose: 30 mg / kg / day (0.118 mmol / kg / day) * If D) is not NR, the daily dose range is 0.0196-1.96 mmol / kg.

[0087] Thus, the method of treatment of the second aspect may involve, for example, oral administration of: A) a dose of 0.48 to 24 mmol / kg / day, typically 0.48 to 4.8 mmol / kg / day, preferably 1.8 to 4.8 mmol / kg / day, more preferably 2.9 to 4.6 mmol / kg / day; optionally B) a dose of 0.31 to 3.05 mmol / kg / day, preferably 0.31 to 1.84 mmol / kg / day, more preferably 0.43 to 1.23 mmol / kg / day; Optionally, C) a dose of 0.031 to 1.24 mmol / kg / day, typically 0.031 to 0.620 mmol / kg / day, preferably 0.062 to 0.50 mmol / kg / day, more preferably 0.093 to 0.37 mmol / kg / day; and / or D) A dose of 0.0196 to 1.96 mmol / kg / day, typically 0.020 to 0.39 mmol / kg / day, preferably 0.039 to 0.31 mmol / kg / day, more preferably 0.059 to 0.24 mmol / kg / day, except that when D) is NR, the dose is 0.39 mmol / kg / day or less.

[0088] The daily dosage can be achieved by administering one or more doses to the consumer / patient per day. For example, the patient can drink the above solution or suspension once, twice, or three times a day. Each dose or drink preferably contains 4.78 mmol / kg or less of A).

[0089] The treatment method of the second embodiment can be carried out for a period of 1 to 12 weeks, for example, 2 to 8 weeks, preferably 3 to 8 weeks. If the treatment is carried out for a long period, the risk of side effects increases. A shorter period may not be sufficient for the therapeutic effect.

[0090] In a third aspect of the present disclosure, there is provided a method for treating a condition selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, type 2 diabetes or obesity, comprising oral administration to a subject in need thereof of: A) serine, glycine, betaine, N-acetylglycine, N-acetylserine, dimethylglycine, sarcosine and / or phosphoserine at a dose of 0.48 to 24.8 mmol / kg / day, such as 0.48 to 4.8 mmol / kg / day, for example 1.8 to 4.8 mmol / kg / day, for example 2.9 to 4.6 mmol / kg / day; B) optionally N-acetylcysteine, cysteine and / or cystine, at a dose of 0.31 to 3.05 mmol / kg / day, for example 0.31 to 1.84 mmol / kg / day, for example 0.43 to 1.23 mmol / kg / day; C) optionally carnitine, deoxycarnitine, gamma-butyrobetaine, 4-trimethylammoniobutanal, 3-hydroxy-N6,N6,N6-trimethyl-L-lysine, N6,N6,N6-trimethyl-L-lysine and / or lysine, at a dose of 0.031 to 0.24 mmol / kg / day, such as 0.031 to 0.620 mmol / kg / day, for example 0.062 to 0.50 mmol / kg / day, for example 0.093 to 0.37 mmol / kg / day; D) nicotinamide riboside (NR), quinolinate, deamino-NAD+, nicotinate D-ribonucleotide, nicotinamide D-ribonucleotide, nicotinate D-ribonucleoside, nicotinamide and / or nicotinate, for example, at a dose of 0.0196 to 1.96 mmol / kg / day, for example, 0.020 to 0.39 mmol / kg / day, for example, 0.039 to 0.31 mmol / kg / day, for example, 0.059 to 0.24 mmol / kg / day, D) If NR, the dose must be 0.39 mmol / kg / day or less.

[0091] The embodiments and examples of the first and second aspects also apply to the third aspect mutatis mutandis.

[0092] It is not necessary for the patient / consumer to take the substances of the present disclosure at the same time: the therapeutic effect may also be achieved if the substances are taken separately or sequentially, preferably within one day, and more preferably within one hour.

[0093] In a fourth aspect of the present disclosure, there is provided a substance for simultaneous, separate or sequential use in a therapeutic method of treating a subject, comprising: A) serine, glycine, betaine, N-acetylglycine, N-acetylserine, dimethylglycine, sarcosine and / or phosphoserine; B) optionally N-acetylcysteine, cysteine and / or cysteine; C) optionally carnitine, deoxycarnitine, γ-butyrobetaine, 4-trimethylammoniobutanal, 3-hydroxy-N6,N6,N6-trimethyl-L-lysine, N6,N6,N6-trimethyl-L-lysine and / or lysine; and D) Nicotinamide riboside, quinolinate, deamino-NAD+, nicotinate D-ribonucleotide, nicotinamide D-ribonucleotide, nicotinate D-ribonucleoside, nicotinamide and / or nicotinate.

[0094] A fourth embodiment may be a combined formulation of two or more units, such as, for example, a first unit comprising A), a second unit comprising D), optionally a third unit comprising B), and optionally a fourth unit comprising C).

[0095] The embodiments and examples of the first and second aspects apply mutatis mutandis to the third and fourth aspects. [Example]

[0096] experiment Experimental procedure subject To study the liver response to HS, 86 subjects with varying degrees of HS were recruited. The clinical characteristics of the subjects are shown in Table 1. Liver tissue samples were also collected from 12 morbidly obese subjects who underwent bariatric surgery. The characteristics of the morbidly obese subjects are shown in Table 2. The mRNA expression of the identified target genes was measured in the livers of obese and healthy subjects. To demonstrate the effects of serine on the liver, six additional subjects were recruited. The characteristics of the subjects before and after serine supplementation are shown in Table 3. The subjects included in this study met all criteria for NAFLD, including the exclusion of other chronic liver diseases such as viral hepatitis, hazardous alcohol intake, and metabolic disorders (e.g., hemochromatosis).

[0097] Table 1. Clinical characteristics of the 86 study participants. Data are presented as mean ± SD. P values indicate the level of significance between subjects with low and high fatty liver (HS). [Table 2]

[0098] Table 2. Clinical characteristics of 12 obese subjects undergoing bariatric surgery with high HS. Data are presented as mean ± SD. [Table 3]

[0099] Table 3. Clinical characteristics of the six subjects involved in the serine supplementation study. Data are presented as mean ± SD. P values indicate significant differences before and after oral serine supplementation. [Table 4]

[0100] Measurement of liver fat, subcutaneous fat, and intraperitoneal fat Magnetic resonance experiments were performed using three 1.5T clinical imaging devices (1x Sonata and 2x Avanto, Siemens, Erlangen, Germany). Liver fat content was determined using proton magnetic resonance spectroscopy, and subcutaneous abdominal and visceral fat was measured by magnetic resonance imaging ( Adiels et al., 2006 ; Lundbom et al., 2011 ).

[0101] Flux data measurement Lipoprotein fluxes were measured in 73 fasting subjects using stable isotope infusion. After a bolus infusion of d3-leucine and d5-glycerol, large (VLDL1) and small (VLDL2) VLDL subfractions were isolated by ultracentrifugation, and plasma free leucine, leucine in apoB, and glycerol enrichment in TG were measured using gas chromatography-mass spectrometry (Adiels et al., 2005). Metabolic fluxes were calculated using mathematical modeling as previously described (Adiels et al., 2005).

[0102] Calculating muscle mass and body fat Each subject's muscle mass was calculated from their lean mass using a previously described relationship (Clark et al., 2014) based on their body fat mass. To predict the deficit in body fat mass in the remaining 29 subjects, a linear equation was fitted between BMI and the body fat mass of 44 of the subjects. The linear equation was defined as follows: fat mass (kg) = 1.763 * BMI - 26.75 (R^2 = 0.69). Using this equation, body fat mass was calculated for the 29 subjects, and then lean mass was calculated by subtracting body fat mass from the subjects' body weight. Finally, each subject's muscle mass was calculated based on the previously derived equation (Clark et al., 2014): muscle mass = 0.63 * lean mass - 4.1.

[0103] Input and output of liver GEM during fasting During fasting, the liver consumes gluconeogenic substrates, nonesterified FAs and AAs, to produce blood glucose (as an energy substrate for the brain), VLDL (as an energy substrate for the rest of the body), ketone bodies, and plasma proteins. Proteins secreted by the liver (mainly albumin) are not necessarily a net loss to the liver because proteins can be recycled. However, in this study, urinary urea loss was used as a proxy for net protein loss from the liver.

[0104] Thus, the input variables of the model are i) AA, ii) lactate, and iii) FA and glycerol. The output variables are iv) glucose from gluconeogenesis and glycogenolysis, v) ketone bodies, and measured VLDL secretion.

[0105] i)AA In the fasting state, some AA is released by muscle tissue. Pozefsky et al. (Pozefsky et al., 1976) experimentally quantified AA release from muscle tissue in the fasting state. They found that approximately 60% of the total AA released from muscle were glutamine and alanine, which are the major substrates used for hepatic gluconeogenesis. These experimentally determined values were incorporated into a model based on each subject's muscle mass.

[0106] Adipose tissue also releases AA into the bloodstream. Because the subjects in this study had varying degrees of obesity, it is important to know whether AA release differs between lean and obese subjects. Patterson et al. (Patterson et al., 2002) found that AA release is proportional to the amount of adipose tissue a person has, but it also depends on blood flow, which decreases as the amount of adipose tissue increases. Therefore, AA release from adipose tissue is independent of obesity. Therefore, an additional input of AA based on adipose tissue mass was included in the model. This contribution was calculated based on the work of Frayn and Karpe (Frayn & Karpe, 2014), who measured the blood volume entering and leaving adipose tissue (3-4 ml / min, 100 g adipose tissue).

[0107] Another method (Ardilouze et al., 2004) provided information on human body fat mass based on BMI, sex, and age according to the following formula: Percent body fat = (1.2 * BMI) + (0.23 * age) - (10.8 * sex) - 5.4, where sex is 0 females and 1 male (Deurenberg et al., 1991). This resulted in an average body fat mass of approximately 15 kg and an average blood flow through adipose tissue of approximately 31.5 L / h. Patterson et al. (Patterson et al., 2002) provided values for AA release (μmol / L) based on body fat, so AA release by adipose tissue (mmol / h) was calculated for each subject and used as input to the individualized model.

[0108] Muscle and adipose tissues are not the only sources of AA for the liver during fasting. It has been shown that rat liver catabolizes approximately 25% of total intracellular protein during the first 24 hours of fasting (Cuervo & Dice, 1996). In the present analysis, the total AA released from muscle and adipose tissue does not appear to satisfy the liver's demand for AA. During a 16-hour fast, the urea excretion rate measured in humans was 392 ± 44 mmol urea / 24 hours (Norrelund et al., 2001). Assuming an average nitrogen content of 1.45 nitrogen atoms per AA and an average AA molar mass of 136.5 g / mol, liver AA consumption after a 16-hour fast was therefore, on average, close to 80 g / day (392 mmol / 24 hours * 136.5 g / mol / 1000 / 1.45 = 77.6 g AA / day). This value remained nearly constant after 40 h of fasting (440 mmol / 24 h), indicating a maintenance (or even an increase) of AA consumption in the liver during fasting. The total amount of AA released by muscle and adipose tissue was calculated to be close to 35 g / day, indicating that the liver itself can catabolize a relatively large amount—approximately 40–45 g / day—in this study. The AA composition of the human liver was measured by Benga & Ferdinand (Benga & Ferdinand, 1995). To achieve realistic values for net AA consumption, the molar ratio of AA in the liver was incorporated as an additional input reaction into the model.

[0109] ii) Lactic acid Lactate is used as a substrate for hepatic gluconeogenesis. Wallace (2002) has proposed that, assuming a resting state, the total amount of lactate produced by red blood cells, kidneys, medulla, and retina is approximately 40 g per day. In addition, an extra 40 g is produced from other parts of the body, for a total of approximately 80 g. This corresponds to approximately 3.3 g / h = 37 mmol / h, which was used as input into the model.

[0110] iii) FA and glycerol FAs are used by the liver to produce TGs in VLDL. Glycerol, a by-product of TG breakdown and subsequent FA release by adipose tissue, can be used as a gluconeogenic substrate. FA and glycerol release from adipose tissue was estimated based on a study by McQuaid et al. (2011), where values for FA and glycerol release from adipose tissue were retrieved for each subject in the fasting state. This average value was approximately 30 μmol / min kg fat mass, which is equivalent to 1.8 mmol / h kg fat mass. Because the molar ratio of glycerol release to FA release was 1:3, glycerol release was set at 0.6 mmol / h kg fat mass. Both of these values were considered upper limits. However, Bickerton et al. (2007) measured total FA influx into muscle in fasting subjects and found that only approximately 4% of the FA released by adipose tissue was taken up by muscle. Therefore, 1.8 mmol / h of released FA was used as input to the model.

[0111] iv) Gluconeogenesis and glycogenolysis Lactate, glutamine, alanine, and glycerol are the main substrates for gluconeogenesis. Another source of glucose is glycogenolysis. McQuaid et al. (2011) and Hellerstein et al. (1997) reported that under normal overnight fasting conditions, the contributions of gluconeogenesis and glycogenolysis to hepatic glucose production are approximately equal. McQuaid et al. (2011) also found that glycogenolysis in humans after an overnight fast was approximately 5.5 μmol / kg / min. This corresponds to an average contribution from glycogenolysis of approximately 5.7 g glucose / h for the subjects in this study. The brain requires approximately 6 g glucose / h during early fasting, when ketone body production is still low (Bourre, 2006). This suggests that the total glucose output from the liver during overnight fasting is on the order of 10–15 g / h, which is significantly higher than 6 g / h. In conclusion, the absolute minimum contribution of gluconeogenesis to glucose production was established as 16.7 mmol / h (3 g / h).

[0112] v) Ketone bodies Total ketone body production in obese humans increases dramatically after 2–3 days of fasting, up to approximately 60 mmol / h, and after 17–24 days of fasting, up to approximately 75 mmol / h (Reichard et al., 1974). However, during overnight fasting, glycogenolysis should meet the majority of brain energy needs, and therefore the ketone body production rates of acetoacetate and β-hydroxybutyrate were set at the lower limit of 0.1 mmol / h in the model.

[0113] A personalized genome-scale metabolic model for liver tissue A functional GEM for hepatocytes in the liver, iHepatocytes2322, was reconstructed based on hepatocyte-specific proteomic data from the Human Protein Atlas (HPA, http: / / www.proteinatlas.org) (Uhlen et al., 2015). Using iHepatocytes2322 in combination with flux balance analysis, in silico liver metabolic simulations were performed for each subject participating in the study. Measured / calculated uptake and secretion rates of key metabolites were incorporated into each GEM to predict intracellular liver fluxes for each patient. During the personalized simulation of liver tissue GEMs, the model was allowed to uptake oxygen, phosphate, minerals, etc., while the uptake of other metabolites was blocked to simulate a fasting state. After setting all boundaries, fluxes for all subjects were calculated by minimizing the sum of fluxes, based on the assumption that cells minimize pathway usage for economic reasons. To test the robustness of the results, fluxes were also calculated by random sampling without minimizing the sum of fluxes, and the same significant results were observed.

[0114] To investigate the contribution of personalized inputs and outputs (FA uptake and VLDL secretion) to our conclusions, we performed a randomized control analysis (random values across the range of maximum and minimum values for all patients). Using random FA uptake or VLDL secretion alone as input or output to the personalized model, we found that the correlation between NNT and the responses induced by GSR and HS was significantly reduced. Furthermore, when both random FA uptake and VLDL secretion were used, the correlation became non-significant. Thus, we concluded that both personalized inputs and outputs drove the conclusions reached in the study.

[0115] Metabolomics Data Detection and quantification of untargeted metabolites was performed by metabolomics provider Metabolon Inc. (Durham, USA) on fasting plasma samples collected from subjects with varying degrees of HS. Samples were prepared using an automated MicroLab STAR® system from Hamilton Company. For quality control purposes, a recovery standard was added before the first step of the extraction process. To remove proteins and dissociated small molecules bound to proteins or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, proteins were precipitated with methanol (Glen Mills GenoGrinder 2000) with vigorous shaking for 2 minutes, followed by centrifugation. The resulting extract was divided into four fractions: one for analysis by UPLC-MS / MS using positive-ion mode electrospray ionization, one for analysis by UPLC-MS / MS using negative-ion mode electrospray ionization, one for analysis by GC-MS, and one sample was kept for backup.

[0116] After logarithmic transformation, Welch's two-sample t-test was used to identify metabolites that differed significantly between high and low HS subjects, with the minimum observed values for each compound. P values were corrected for multiple testing. During the identification of significant and significantly correlated metabolites, data were not imputed for missing values. Correlation analysis between metabolites was performed if both metabolites were detected in at least 30 subjects involved in the study.

[0117] Mouse experiments Twenty male C57BL / 6N mice were fed standard mouse chow (Purina 7012, Harlan Teklad) and housed under a 12-hour light / dark cycle. From 8 weeks of age, mice were fed a Western diet (TD.88137, Harlan Laboratories, WI, USA) for 14 days. They were then divided into two groups of 10 mice. One group of mice was fed a Western diet supplemented with NR (400 mg / kg) and serine (300 mg / kg) by oral gavage once a day and NAC (1 g / L) in drinking water for 14 days. The other group was fed only a Western diet for 14 days. All procedures were approved by the local animal ethics committee and were performed in accordance with the mandatory guidelines.

[0118] Lipid extraction and analysis Lipids were extracted as previously described (Lofgren et al., 2012). An internal standard was added during extraction. Lipids were analyzed using a combination of HPLC and mass spectrometry, as described (Stahlman et al., 2013). Briefly, ceramides (CER) were purified using linear-phase HPLC. Cholesteryl esters (CE), triacylglycerols (TAG), phosphatidylethanolamines (PE), phosphatidylcholines (PC), and sphingomyelins (SM) were quantified using a QTRAP5500 mass spectrometer (Sciex, Concord, Canada) equipped with a robotic nanoflow ion source, TriVersa NanoMate (Advion BioSciences, Ithaca, NJ). CER was analyzed using reversed-phase HPLC coupled to a triple quadrupole Quattro Premier mass spectrometer (Waters, Milford, MA, USA).

[0119] Human trials: serine supplementation The effects of short-term dietary supplementation with serine on HS and fasting plasma marker levels of liver function were evaluated in six subjects with high HS. The characteristics of the six subjects before and after supplementation are shown in Table 3. Each patient received a single oral dose of ~20 g L-serine per day (200 mg / kg) for 14 days.

[0120] result Characteristics of subjects with different degrees of HS Eighty-six subjects (75 men and 11 women) were recruited, and their liver fat content was determined using magnetic resonance spectroscopy (Adiels et al., 2006; Lundbom et al., 2011). Pearson correlation coefficients (r) between HS and other clinical parameters were calculated, and HS was significantly (P < 0.05) positively correlated with body weight, body mass index (BMI), insulin resistance (HOMA-IR), plasma triglycerides (TG), and the liver enzyme alanine aminotransferase (ALT) level (Figure 1A). The ALT-to-aspartate transaminase (AST) ratio was also significantly (P < 0.05) correlated with HS (r = 0.57). Neither other liver-related clinical parameters (AST, alkaline phosphatase (ALP), and gamma-glutamyltransferase (μGT)), blood lipid-related parameters (high-density lipoprotein (HDL) cholesterol, total cholesterol, and apolipoprotein B (apoB)), nor the inflammatory marker C-reactive protein (CRP) were significantly correlated with HS.

[0121] Subjects with varying degrees of HS were classified into two groups of 43 subjects based on their liver fat percentage: high HS (>5.5%) and low HS (<5.5%) (Table 1). Subjects with high HS were found to be significantly heavier (P < 0.05) with a greater BMI. Fasting plasma glucose and fasting plasma insulin (FPI) concentrations were significantly higher in subjects with high HS compared with subjects with low HS (P < 0.05) (Figure 1B). Mean plasma triglyceride concentrations were 2.05 mmol / L and 1.67 mmol / L in subjects with high HS and low HS, respectively (Figure 1B). No significant plasma differences were detected in other lipid parameters, including apoB, HDL cholesterol, and total cholesterol (Table 1). ALT levels were significantly higher in subjects with high HS (Figure 1B). In summary, the average low HS subject involved in this study was overweight, borderline hypertriglyceridemic but insulin sensitive, whereas the average high HS subject was obese, hypertriglyceridemic and insulin resistant but did not have T2D.

[0122] Individualized liver tissue GEM To elucidate the molecular mechanisms underlying HS, we employed constraint-based modeling techniques to identify key hepatic metabolic changes among subjects with varying degrees of HS. The secretion rates of non-esterified fatty acids (FAs) and amino acids (AAs) from adipose and muscle tissues were calculated based on each subject's body composition and used as inputs to the personalized liver GEM, along with lactate secreted by erythrocytes. Because the level of TG-rich very-low-density lipoprotein (VLDL) is a major determinant of plasma TG, we combined kinetic studies using stable isotopes and multicompartment modeling to estimate VLDL kinetic parameters in 73 subjects (65 men and 8 women) who participated in the study. A significant correlation (r = 0.581, P < 0.001) was observed between secreted VLDL and HS, and the VLDL secretion rate was used as the objective function for the personalized liver GEM.

[0123] The desired dynamics of hepatic metabolism in response to increased HS were simulated using inputs and outputs as constraints for the personalized GEM. The intracellular fluxes in the liver for each subject were predicted, and the Pearson correlation coefficient between the intracellular fluxes and HS for each subject was calculated. Reactions involved in protein synthesis were found to have the highest correlation with HS (r = 0.57, P < 0.001). The apoB content in total VLDL produced by the liver was also quantified, and it was found to be significantly correlated with the measured HS (r = 0.581, P < 0.001) (Figure 2A). This correlation was very similar to that observed between the TG content in total VLDL produced and the measured HS (r = 0.576, P < 0.001) (Figure 2B). Therefore, it was observed that personalized GEMs can predict the hepatic response to increased HS.

[0124] The reactions with the second and third highest correlations with HS were those involving the reduction of HO (r = 0.482, P < 0.001) and those associated with nicotinamide nucleotide transhydrogenase (NNT) (r = 0.479, P < 0.001), respectively. NNT catalyzes the interconversion of NADH and NADP to NAD and NADPH in mitochondria. NADPH is used to regenerate glutathione (GSH) through the reduction of glutathione disulfide (GSSG) catalyzed by glutathione reductase (GSR). Therefore, NNT plays an important role in providing NAD for fat oxidation and NADPH for redox detoxification. In particular, the flux carried by reactions associated with GSR was found to be one of the most highly correlated with HS (r = 0.478, P < 0.001). Furthermore, reactions involved in fat oxidation were found to be significantly correlated with HS (r = 0.477, P < 0.001). Increased fluxes catalyzed by NNT and GSR-catalyzed reactions generate additional NAD+, which is necessary for increased fat oxidation, and GSH, which is necessary for scavenging excess reactive oxygen species resulting from increased fat oxidation. It has previously been reported that NNT is essential for normal cellular metabolism and mitochondrial defense against oxidative stress (Huang et al., 2006). Furthermore, a significant correlation was observed between HS and secreted ketone bodies, which are one of the major products of hepatic GEM (r = 0.475, P < 0.001).

[0125] HS results from an imbalance between de novo synthesis, oxidation, uptake, and export of FAs (Tamura & Shimomura, 2005). Therefore, we calculated the difference between the rates of FA uptake and secretion in the liver of each subject, defined as net fat influx (NFI), and calculated the correlation between intracellular flux and NFI. In particular, the reactions catalyzed by GSR (r = 0.812, P < 0.001) and NNT (r = 0.811, P < 0.001) were found to have the highest correlation with NFI. Reactions catalyzed by glutathione peroxidase (GPX) and peroxiredoxin (PRDX), which detoxify superoxides and hydroperoxides, were also found to be significantly correlated with NFI (r = 0.812, P < 0.001). Furthermore, a significant correlation was observed between NFI and secreted ketone bodies (r = 0.782, P < 0.001).

[0126] In silico analysis showed that, theoretically, increased HS could be compensated for by increased flux brought about by reactions catalyzed by NNT, GSR, GPX, and PRDX. However, the demand for increased flux is not met in practice, leading to increased HS in NAFLD patients. Given that the simulation demonstrated an ideal hepatic response to increased HS, upregulating fat oxidation and increasing GSH availability, this may provide a therapeutic strategy for NAFLD subjects.

[0127] Glycine is the limiting substrate for de novo synthesis of GSH in NAFLD GSH depletion can lead to mitochondrial dysfunction and cell death (Fernandez-Checa & Kaplowitz, 2005; Garcia-Canaveras et al., 2011). Based on in silico analysis, it has been proposed that increased expression of NNT can increase NAD+ levels due to increased fat oxidation, while NNT and GSR can increase GSH levels, which are necessary to resist oxidative stress and maintain a reducing environment in the liver. However, NNT and GSR expression cannot be continuously increased in vivo, which leads to NAD+ and GSH depletion and ultimately to fat accumulation in the liver. Indeed, hepatic depletion of NAD+ has been reported in mouse models of NAFLD (Gariani et al., 2016; Zhou et al., 2016). Furthermore, lower concentrations of both GSH and GSSG, as well as a decreased GSH / GSSG ratio, have been reported in the liver (Garcia-Canaveras et al., 2011) and serum (Kalhan et al., 2011) of NAFLD patients compared with healthy subjects.

[0128] Depleted GSH can also be replaced by de novo synthesis of GSH from glutamine, glycine, and cysteine, which can be taken up from plasma. To detect the plasma levels of these AAs, we performed untargeted metabolomic profiling in plasma from 86 subjects and analyzed the levels of ~520 metabolites. Correlations between plasma metabolite levels and HS were evaluated. Fasting plasma levels of glycine and N-acetylglycine, as well as betaine and serine (which can be converted to glycine), showed a significant negative correlation with HS. Correlation coefficients between plasma metabolites significantly correlated with HS were also evaluated, and plasma glycine levels were found to be most highly correlated with plasma serine levels among all other measured metabolites (r = 0.77, P < 0.05). It should be noted that no significant correlation was detected between HS and plasma levels of cysteine and glutamine (which are also required for the de novo synthesis of GSH).

[0129] We also investigated whether any of the plasma metabolites showed significant differences between the two groups of subjects divided according to their HS levels. We found that the levels of glycine, serine, betaine, and N-acetylglycine were significantly (Welsh's t-test, P < 0.05) lower in high-HS subjects compared with low-HS subjects (Figure 3). In addition to glycine-related metabolites, we also found that the levels of butyrylcarnitine, glycylphenylalanine, gamma-tocopherol (vitamin E), kynurenate, N-delta-acetylornithine, N-methylproline, and numerous lipid structures, which have been shown to correlate with HS, were significantly altered between high- and low-HS subjects (Welsh's t-test, P < 0.05) (Figure 3).

[0130] Decreased expression of enzymes involved in GSH formation The crucial role of GSH metabolism in the development of NAFLD has been revealed. In this context, we compared the expression of enzymes involved in NNT, GSR, and de novo GSH synthesis in human liver samples obtained from a separate cohort of 12 obese subjects with high HS who underwent bariatric surgery (Table 2) with liver samples obtained from seven healthy individuals (previously described in (Uhlen et al., 2015)). The mRNA expression of the rate-limiting enzymes in NNT, GSR, and de novo GSH synthesis, namely glutamate cysteine ligase, catalytic subunit (GCLC) and glutamate cysteine ligase, modifying subunit (GCLM), was significantly lower in livers from obese subjects than from healthy controls (Figure 4). This indicates that decreased expression of NNT and GSR may increase HS, which is consistent with the results of individualized modeling of subjects with varying degrees of HS.

[0131] Supplementation of GSH and NAD+ precursors reduces HS in mice Analyses have demonstrated depletion of NAD+ and GSH in subjects with high HS. Supplementation with natural NAD+ precursors, such as nicotinamide riboside (NR), tryptophan, niacin, and nicotinamide, has been shown to increase NAD+ levels in vivo (Canto et al., 2012; Houtkooper et al., 2010). Plasma and hepatic concentrations of GSH are depleted in NAFLD patients and cannot be increased by GSH supplementation. Instead, GSH must be synthesized de novo or via a rescue pathway in the liver. Analyses suggest that in subjects with high fasting HS due to glycine depletion, GSH levels are insufficient to maintain and regulate the hepatic thiol redox state. Glycine can be synthesized through serine interconversion via serine hydroxymethyltransferase, with the simultaneous conversion of tetrahydrofolate (THF) to 5,10-methylene-THF. During the conversion of serine to glycine, an additional carbon unit is provided for one-carbon metabolism. Taken together, it has been hypothesized that dietary supplementation with NR increases the levels of NAD+, which is necessary for increased fat oxidation, and serine increases the levels of glycine and GSH (due to intracellular GSH synthesis from glycine). Supplementing the substrates for NAD+ and GSH may increase the amount of fat oxidized in the liver, reduce oxidative stress resulting from increased fat oxidation, decrease the levels of HS, and ultimately improve liver function.

[0132] To evaluate the effects of GSH and NAD+ supplementation on the development of HS in mice, a cocktail containing serine, N-acetyl-L-cysteine (NAC), and NR was supplemented to mice fed a Western diet containing high levels of fat and sucrose. Serine was included in the cocktail because it can be readily converted to glycine, while NAC was included because cysteine may be the limiting metabolite in GSH synthesis after glycine supplementation. NR was included in the cocktail to increase the amount of NAD+ in the liver. Male C57BL / 6N mice fed a Western diet were treated with 300 mg / kg / day of serine and 400 mg / kg / day of NR via gavage and 1 g / L of NAC in drinking water for 14 days. Mice were sacrificed 4 hours after the last treatment. Liver lipidomics analysis was performed and observed the following: a 50% decrease in hepatic TGs (Figure 5a:A), a trend toward decreased cholesterol ester levels (Figure 5a:B) and ceramide levels (Figure 5a:C); a trend toward increased sphingomyelin levels (Figure 5a:D); and no significant changes in phosphatidylethanolamine levels (Figure 5a:E). Glycine and serine levels were also measured, and their plasma levels were found to be significantly increased after cocktail supplementation (Figure 5a:F). Finally, hepatic levels of TGs with different chain lengths were measured, and shorter chain lengths of TGs, which are preferentially oxidized in mitochondria, were found to be significantly decreased after supplementation (Figure 5b:G). Therefore, supplementation with metabolites predicted by personalized modeling was proven to promote hepatic fat oxidation and prevent HS. Thus, mouse studies confirmed the proposed therapeutic strategy for protection against NAFLD progression.

[0133] Serine supplementation reduces HS in humans To confirm the unique contribution of serine supplementation in reducing HS, the effects of short-term dietary serine supplementation on HS and fasting levels of plasma markers of liver function were evaluated in six subjects with high HS. The characteristics of the six subjects before and after supplementation are shown in Table 3. Each patient received a single oral dose of ~20 g of L-serine per day (200 mg / kg) for 14 days. Supplementation was well tolerated by all subjects. Plasma levels of serine were found to increase significantly, while plasma levels of ALT, AST, and ALP were found to decrease significantly after supplementation (Table 3). In particular, plasma levels of ALT (Figure 5c:H) and AST (Figure 5c:I) consistently decreased in all six subjects, and ALP decreased in five of the participating subjects (Figure 5c:J). Furthermore, plasma TG was found to decrease in five study subjects and remain unchanged in the remaining one (Figure 5c:K). HS was also measured using magnetic resonance spectroscopy before and after serine supplementation, demonstrating that HS was significantly reduced after serine supplementation (Table 3). HS was reduced in all six patients, with the relative reduction in NAFLD patients ranging from 1.0 to 23%.

[0134] Calibration Test Nine healthy subjects (BMI < 30) were recruited. Therefore, they did not suffer from T2D or NAFLD and were not taking any medication. All subjects participating in the study signed an informed consent form.

[0135] The subjects stayed in the same hotel and ate the same breakfast and lunch throughout the study, which allowed for monitoring for possible side effects of the medication.

[0136] The study began at 08:00 each day, and supplementation was carried out as follows: On day 1, each subject received a single oral dose of 1 g (0.0039 mol) of NR. On the second day, each subject received a single oral dose of 3 g (0.019 mol) of L-carnitine. On the third day, each subject received a single oral dose of 5 g (0.031 mol) of NAC. On day 4, each subject received one oral dose of the complete medication, in this case 1 g NR, 3 g L-carnitine, 5 g NAC, and 20 g L-serine. On the fifth day, each subject received a single oral dose of 20 g (0.19 mol) of L-serine.

[0137] In the complete drug product, the molar ratio of serine to NR was about 48:1, the molar ratio of serine to NAC was about 6.1:1, and the molar ratio of serine to L-carnitine was about 10:1.

[0138] On days 1, 2, 3 and 5, blood samples were taken before (08:00) and after (12:00) supplementation.

[0139] On the fourth day, blood samples were taken eight times (8:00, 9:00, 10:00, 11:00, 12:00, 13:00, 14:00, and 15:00) (to understand the kinetics of all drug substances).

[0140] A glucose monitoring device was used to measure the subjects' glucose levels during the study.

[0141] Plasma levels of glucose, insulin, gamma GT, bilirubin, ALP, ASAT, ALAT, FFA, TAG, total cholesterol, HDL and LDL were measured before and after supplementation.

[0142] Plasma concentrations of serine, L-carnitine, NAC, and NAD+ were measured using a targeted metabolomics platform.

[0143] No subjects withdrew from the study and no adverse events were reported.

[0144] It has been observed that the plasma serine levels of diseased patients are approximately 50% of those of healthy individuals. Therefore, it was desirable to find an oral serine dose that would result in a one-fold increase in plasma serine levels. Furthermore, a one-fold increase in plasma serine levels was expected to reflect a significant increase in liver serine levels. As discussed in the overview above, cysteine becomes limiting for antioxidant formation after adequate serine (or glycine) supplementation. Therefore, it was also desirable to find an oral NAC dose that would result in a one-fold increase in plasma NAC levels. Finally, following the considerations in the overview above, it was desirable to find an oral L-carnitine dose that would result in a one-fold increase in plasma L-carnitine levels.

[0145] Three-compartment ordinary differential equation (ODE) models representing the stomach, intestine, and blood were developed based on published information. The models were fitted to experimentally measured plasma concentrations. One model each for serine, L-carnitine, and NAC was developed based on the average plasma concentrations of the substance in question in subjects over a period of up to 24 hours after ingestion. The bioavailability of each substance was set according to literature values.

[0146] An interpolation of the plasma concentration of each substance was constructed for each subject. The average of the interpolations was used as the target concentration curve. A model was then fitted to this curve. After fitting the model to each substance, the model was used to predict the plasma concentration achieved when receiving twice-daily supplementation therapy. Individual doses of the substances were adjusted to achieve the desired 100% increase in mean (long-term) plasma concentration without displacing safe doses for human consumption.

[0147] The model predicted that a twice-daily dose of 12.75 g (0.121 mol) serine would produce the desired long-term increase in mean plasma serine concentration of 100%. Such a twice-daily dose would be equivalent to 3.5 mmol / kg / day of serine for a 70 kg patient. Doses up to 400 mg / kg / day (approximately 25-30 g / day) have been tested in humans and shown to be safe.

[0148] For L-carnitine, the model predicted that a twice-daily dose of 8.2 g (0.0509 mol) of L-carnitine would produce the desired long-term increase in mean plasma L-carnitine concentrations by 100%. However, because no long-term supplementation studies have examined the safety of L-carnitine doses greater than 7 g (0.0434 mol) per day, the recommended dose was lowered to 3 g (0.0186 mol) twice-daily. This resulted in a long-term increase in mean plasma concentrations of 37%, which was considered a reasonable trade-off between the risk of toxicity and increased plasma concentrations. A twice-daily dose of 3 g of L-carnitine corresponds to 0.53 mmol / kg / day of L-carnitine in a 70 kg patient.

[0149] For NAC, the model predicted that a twice-daily dose of 3.2 g (0.0196 mol) of NAC would produce the desired long-term increase in mean plasma NAC concentration of 100%. Such a twice-daily dose would be equivalent to 0.56 mmol / kg / day of serine for a 70 kg patient. Daily doses of 4 to 6 grams of NAC have been shown to be safe in humans.

[0150] (2016) support the continued use of NR at a dose of 1 g (0.0039 mol) twice daily. Such a twice-daily dose corresponds to 0.11 mmol / kg / day of NR in a 70 kg patient.

[0151] In the prepared complete drug, the molar ratio of serine to NR was about 31:1, the molar ratio of serine to NAC was about 6.2:1, and the molar ratio of serine to L-carnitine was about 6.5:1.

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Claims

1. A composition comprising: A) serine; B) N-acetylcysteine; C) carnitine; and D) nicotinamide riboside or nicotinamide, wherein the molar ratio of A) to D) is from 250:1 to 1.5:1; the molar ratio of A) to B) is from 16:1 to 1:4, The substances included in groups A) to D) comprise at least 25% of the dry weight of the composition.

2. the molar ratio of A) to B) is from 12:1 to 1.5:1; and / or the molar ratio of A) to C) is from 150:1 to 1:1, and / or the molar ratio of A) to D) is 150:1 to 3:1; The composition of claim 1.

3. 3. The composition of claim 1 or 2, wherein A) is L-serine, and / or C) is L-carnitine, and / or D) is nicotinamide riboside.

4. The composition according to any one of claims 1 to 3, which is an aqueous solution or suspension.

5. 5. The composition according to claim 4, wherein the concentration of A) is 0.20 to 2.4 mmol / ml and / or the concentration of D) is 0.006 to 0.12 mmol / ml.

6. The composition according to any one of claims 1 to 3, which is a solid.

7. 7. The composition of claim 6, which is a solid powder.

8. 8. The composition of claim 7, wherein the powder is packaged and the pack of powder comprises 48 to 478 mmol of A) and / or 2.0 to 39.2 mmol of D) if D) is nicotinamide riboside (NR), or 2.0 to 196 mmol of D) if D) is not NR.

9. The composition according to any one of claims 1 to 8, wherein the composition is for oral ingestion of: A) doses of 0.48 to 24 mmol / kg / day; B) a dose of 0.31 to 3.05 mmol / kg / day; C) a dose of 0.031 to 1.24 mmol / kg / day; and D) A dose of 0.020 to 0.39 mmol / kg / day.

10. A pharmaceutical composition comprising: A) serine; B) N-acetylcysteine; C) carnitine; and D) nicotinamide riboside or nicotinamide, wherein the molar ratio of A) to D) is from 250:1 to 1.5:1; the molar ratio of A) to B) is from 16:1 to 1:4, The substances included in groups A) to D) comprise at least 25% of the dry weight of said pharmaceutical composition.

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