KR102280261B1 - Method for diagnosing liver disease and by using metabolomics - Google Patents

Abstract

The present invention includes a method for providing information for the diagnosis of liver disease using a specific metabolite, a screening method for a drug for preventing, improving, or treating liver disease, and a quantification agent or quantitative device for a specific metabolite, The present invention relates to a kit for diagnosing liver disease, which uses a sample isolated from an individual, so it is non-invasive and simple to diagnose liver disease, and it has excellent diagnostic accuracy because it can examine various types of metabolites.

Description

Method for diagnosing liver disease using metabolite analysis {Method for diagnosing liver disease and by using metabolomics}

The present invention includes a method for providing information for the diagnosis of liver disease using a specific metabolite, a screening method for a drug for preventing, improving, or treating liver disease, and a quantification agent or quantitative device for a specific metabolite, It relates to a kit for diagnosing liver disease.

Non-alcoholic fatty liver disease (NAFLD) is a fatty liver disease that occurs without significant alcohol intake or drug intake, and generally has a good course. However, some patients with NAFLD may progress to serious liver diseases such as nonalcoholic steatohepatitis, nonalcoholic fatty liver-associated cirrhosis and liver cancer. The prevalence of viral hepatitis, which accounts for a large portion of liver disease, is expected to gradually decrease due to the effects of hepatitis vaccination and antiviral treatment, while the prevalence of nonalcoholic fatty liver disease increases further in line with the increase in metabolic diseases such as obesity, known as risk factors. expected to do

NAFLD includes liver disease that progresses to simple steatosis, nonalcoholic steatohepatitis (NASH), and nonalcoholic fatty liver-associated cirrhosis without an inflammatory or fibrotic response. Since the degree of progression of steatohepatitis and fibrosis greatly affects the prognosis of the disease, the best method (gold standard) for judging the progression of these diseases is a liver biopsy. However, liver biopsy is invasive and has disadvantages such as limited sampling error due to local evaluation and high cost, so efforts are being made to improve the diagnosis rate by non-invasive methods using various biomarkers and imaging tools.

Non-invasive, non-imaging methods reported to date as a diagnostic model of NAFLD include fat liver index (FLI), SteatoTest, NAFLD liver fat score, and hepatic steatosis index (HIS). However, these methods require additional validation through other studies. In addition, SteatoTest is not only NAFLD, hepatitis C, alcoholic fatty liver patients as well as hepatitis C, hepatitis C, alcoholic fatty liver, the model was built, and the formula is not publicly available, so it is difficult to use it in general, and FLI and HIS have two cut-offs. There is a limitation that it is not easy to judge the intermediate) section.

Therefore, there is a need for the development of a technology capable of diagnosing liver disease in a clear, non-invasive and non-imaging manner.

Republic of Korea Patent Publication No. 10-2013-0113046

An object of the present invention is to measure a metabolite concentration in a biological sample isolated from a subject and a normal individual; and

An object of the present invention is to provide a method of providing information for diagnosing liver disease, comprising comparing the metabolite concentration in the subject sample measured in the above step with the metabolite concentration in the normal subject sample.

Another object of the present invention is to measure a metabolite concentration in a biological sample isolated from a subject to which a drug candidate is administered; and

To provide a screening method for a drug for preventing, improving, or treating liver disease, comprising comparing the concentration of the metabolite measured in the above step with the concentration of the corresponding metabolite in a control sample.

Another object of the present invention is to provide a kit for diagnosing liver disease, comprising a quantifying agent or quantifying device for metabolites in a biological sample isolated from a mammal.

Accordingly, the inventors of the present application conducted metabolite analysis on samples separated from patients with epilepsy (or non-alcoholic fatty liver disease) and non-disease patients with improved liver disease (or non-alcoholic fatty liver disease). As a result, 25 types of metabolites The present invention was completed by confirming a significant change in , and confirming that the 25 types of metabolites selected above can be used as biomarkers for diagnosing liver disease.

25 metabolites that can be used as biomarkers for diagnosing liver disease are glycine, glyceric acid, serine, Threonine, proline, β-alanine (β) -alanine), phenylalanine, pyroglutamic acid, α-ketoglutaric acid, glutamic acid, citric acid, decanoic acid, myristic Myristic acid, Palmitoleic acid, Palmitic acid, Linoleic acid, Oleic acid, Stearic acid, Oleamide, Oleani Tril (Oleanitrile), monopalmitin (Monopalmitin), monostearin (Monostearin), α-hydroxyisobutyric acid (α-hydroxyisobutyric acid), glycerol (Glycerol), and / or phosphoric acid (Phosphoric acid).

As used herein, 'diagnosis' refers to determining a subject's susceptibility to a specific disease or disorder, determining whether the subject currently has a specific disease or disorder (eg, liver disease), a specific Determining the prognosis of a subject afflicted with a disease or condition (e.g., liver disease), or monitoring the condition of a subject in order to provide information about the efficacy of treatment (e.g., therametrics) things), etc.

As used herein, the term 'metabolite' refers to a metabolite obtained from a biological sample of biological origin, and may include substances produced by metabolism and metabolic processes or substances generated by chemical metabolism by biological enzymes and molecules.

In the present specification, a 'biological sample' is isolated from a subject or a normal individual and includes tissues, cells, blood, plasma, peritoneal fluid, synovial fluid, saliva, urine, feces, etc., and specifically may be plasma separated from blood. have.

Hereinafter, the present invention will be described in more detail.

In one aspect, glycine, glyceric acid, serine, Threonine, proline, β-alanine from a biological sample isolated from a subject and a normal individual ), Phenylalanine, Pyroglutamic acid, α-ketoglutaric acid, Glutamic acid, Citric acid, Decanoic acid, Myristic acid ( Myristic acid, palmitoleic acid, palmitic acid, linoleic acid, oleic acid, stearic acid, oleamide, oleanithrile ( Oleanitrile), monopalmitin (Monopalmitin), monostearin (Monostearin), α-hydroxyisobutyric acid (α-hydroxyisobutyric acid), glycerol (Glycerol), and one or more metabolites selected from the group consisting of phosphoric acid (Phosphoric acid) measuring the concentration; and

It provides a method of providing information necessary for diagnosing liver disease, comprising comparing the metabolite concentration in the subject sample measured in the above step with the metabolite concentration in the normal subject sample.

As used herein, a 'normal individual' refers to an individual who does not have liver disease among individuals of the same sex, age, and age as the subject.

As used herein, a 'subject sample' refers to a biological sample isolated from a subject, and a 'normal individual sample' refers to a biological sample isolated from a normal subject.

According to one embodiment, the biological sample may be blood or plasma.

According to one embodiment, the blood origin is whole blood, and the whole blood can be pretreated (eg, filtration, distillation, extraction, separation, concentration, inactivation of interfering components, addition of reagents, etc.) to detect metabolites. .

In the present specification, comparing the metabolite concentration in a subject sample with the metabolite concentration in a normal subject sample means comparing the metabolite concentration of the same type. For example, it may be comparing the glycine concentration in the plasma of a subject with the glycine concentration in the plasma of a normal individual.

According to one embodiment, liver disease may be diagnosed by comparing each of the metabolites individually, and as the number of metabolites to be compared increases, the accuracy of diagnosis may be improved.

According to one embodiment, the method of providing information for the treatment of a liver disease is when the metabolite concentration in the subject sample is significantly increased than the metabolite concentration in the normal subject sample, the subject has a liver disease or the subject's liver It can be diagnosed as having a high risk of developing the disease.

According to one embodiment, the method of providing information for the treatment of liver disease is glycine, glyceric acid, serine, threonine, proline, β-alanine ( β-alanine), phenylalanine, pyroglutamic acid, α-ketoglutaric acid, glutamic acid, citric acid, decanoic acid, mi Myristic acid, palmitoleic acid, palmitic acid, linoleic acid, oleic acid, stearic acid, oleamide, oleic acid One selected from the group consisting of nitrile (Oleanitrile), monopalmitin (Monopalmitin), monostearin (Monostearin), α-hydroxyisobutyric acid, glycerol (Glycerol), and phosphoric acid (Phosphoric acid) When the metabolite concentration is measurably significantly increased in a subject sample compared to a normal subject sample (eg, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, or 70% or more), it can be diagnosed that the subject has liver disease or that the subject has a high risk of developing liver disease.

In the present specification, a significantly increased or decreased metabolite concentration means a statistically significant increase or decrease, for example, a p value of less than 0.05 (p<0.05) or less than 0.01 (p<0.05) or less than 0.01 in statistical analysis ( p<0.01) can be said to be statistically significant.

According to one embodiment, the liver disease may be non-alcoholic fatty liver disease (NAFLD).

In the present specification, non-alcoholic fatty liver disease (NAFLD) is a disease that shows fat deposition in the liver in an imaging examination or biopsy without other causes of fat accumulation in the liver, such as alcohol intake or specific drugs, nonalcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH). non-alcoholic fatty liver-associated cirrhosis and the like. Nonalcoholic fatty liver (NAFL) is a case in which fat deposits in the liver but no inflammation and ballooning degeneration, and nonalcoholic steatohepatitis (NASH) is a case in which fat deposits in the liver and inflammation accompanied by ballooning degeneration. It may also be accompanied by fibrosis. Nonalcoholic fatty liver-associated cirrhosis refers to liver cirrhosis with nonalcoholic fatty liver or nonalcoholic steatohepatitis histologically, or cirrhosis occurring in patients with nonalcoholic fatty liver or nonalcoholic steatohepatitis that has been histologically proven.

According to one embodiment, the step of measuring the metabolite concentration may be performed using one or more selected from the group consisting of nuclear magnetic resonance spectroscopy (NMR), chromatography, and chromatography-mass spectrometry. have.

According to one embodiment, the nuclear magnetic resonance spectrometer may be ¹ H-NMR or ¹³ C-NMR, and any nuclear magnetic resonance spectrometer commonly used in the art may be used.

According to one embodiment, the chromatography is gas chromatography (GC), liquid-solid chromatography (Liquid-Solid Chromatography, LSC), paper chromatography (Paper Chromatography, PC), thin layer chromatography (Thin- Layer Chromatography, TLC, Gas-Solid Chromatography, GSC, Liquid-Liquid Chromatography, LLC, Foam Chromatography, FC, Emulsion Chromatography , EC), Gas-Liquid Chromatography (GLC), Ion Chromatography (IC), Gel Filtration Chromatography (GFC) or Gel Permeation Chromatography (GPC) ), and any chromatography commonly used in the art may be used.

According to one embodiment, the mass spectrometer may be a Fourier transform mass spectrometer (FTMS), LTQ-Orbitrap-XL mass spectrometer, MALDI-TOF MS, Q-TOF MS, LTQ-Orbitrap MS, etc., Any mass spectrometer commonly used in the industry may be used.

The chromatography may separate each metabolite using different mobility for each molecule, and the mass spectrometer may identify the metabolite based on structure information as well as molecular weight information of the analyte.

According to one embodiment, the information for the diagnosis of liver disease is at least one selected from the group consisting of liver fat content, ALT (alanine aminotransferase) level, AST (aspartate aminotransferase) level, and GGT (gamma-glutamyl transferase) level. can It is reported that there is a clear correlation between nonalcoholic fatty liver disease and liver fat content, ATL level, AST level, and GGT level.

According to one embodiment, the subject or a normal individual may be a mammal.

According to one embodiment, the mammal may include humans and primates such as monkeys, and rodents such as rats and mice.

According to one embodiment, the subject or a normal individual may be a mammal other than a human.

According to one embodiment, the subject may be a subject suspected of having liver disease.

In one embodiment, in order to simulate a normal individual, a patient with nonalcoholic fatty liver disease ingested pinitol at a dose of 300 to 500 mg/day for 12 weeks, and checked the liver fat content, AST level, ALT level, and GGT level for nonalcoholic fatty liver disease. Improvement of fatty liver disease was confirmed.

The chemical name of pinitol is "2-O-methyl-D-chiro-inositol (3-O-Methyl-D-chiro-inositol)", and has the general formula of Formula 1 below.

[Formula 1]

According to one embodiment, pinitol may be extracted and isolated from natural products or prepared by a conventional organic synthesis method, but is not limited thereto.

Pinitol restores GOT, GPT, and γ-glutamyl transpeptidase (γ-glutamyl transpeptidase) to almost normal levels when taken by patients with fatty liver (Korean Patent Publication No. 10-2004-0084168), and is effective in treating fatty liver disease. it is known

In one embodiment, by analyzing plasma metabolites in non-alcoholic fatty liver disease patients with improved non-alcoholic fatty liver disease after ingestion of pinitol and non-alcoholic fatty liver disease patients before ingesting pinitol, 25 types of plasma increased in non-alcoholic fatty liver disease compared to non-disease patients Metabolites were selected as biomarkers capable of diagnosing liver disease, analyzing prognosis, or confirming improvement in liver disease.

According to one embodiment, the normal subject may be a mammal ingesting pinitol. For example, the normal subject is 100 to 2000 mg/day, 150 to 1800 mg for 1 to 20 weeks, for 5 to 18 weeks, for 8 to 16 weeks, for 10 to 15 weeks, or for 12 weeks. It may be a mammal ingesting pinitol at a dose of /day, 200 to 1500 mg/day, 250 to 1200 mg/day, 300 to 1000 mg/day, 300 to 600 mg/day, or 300 to 500 mg/day.

According to one embodiment, the subject may be a mammal not taking pinitol.

In another aspect, glycine, glyceric acid, serine, threonine, proline, β-alanine (β-) from a biological sample isolated from a subject having liver disease alanine), phenylalanine, pyroglutamic acid, α-ketoglutaric acid, glutamic acid, citric acid, decanoic acid, myristic acid (Myristic acid), palmitoleic acid, palmitic acid, linoleic acid, oleic acid, stearic acid, oleamide, oleanithrile (Oleanitrile), monopalmitin (Monopalmitin), monostearin (Monostearin), α-hydroxyisobutyric acid (α-hydroxyisobutyric acid), glycerol (Glycerol), and phosphoric acid (Phosphoric acid) at least one metabolism selected from the group consisting of It provides a method of providing information for confirming the prognosis of liver disease or the degree of improvement of the liver disease, including the step of measuring the body concentration.

According to one embodiment, in the method of providing information for confirming the prognosis of liver disease or confirming the degree of improvement of liver disease, when the metabolite concentration in the subject sample is significantly reduced than before, the prognosis of liver disease is positive or liver disease This can be considered to be improved, and if the metabolite concentration is increased than before, the prognosis of liver disease may be negative or the liver disease may have worsened.

According to one embodiment, the decrease in the concentration of the metabolite in the subject sample means that 12 months ago, 10 months ago, 8 months ago, 6 months ago, 4 months ago, 3 months ago, 2 months ago, 1 It may be a decrease in the metabolite concentration measured a month ago, 3 weeks ago, 2 weeks ago, or 1 week ago.

According to one embodiment, the positive prognosis of liver disease or improvement of liver disease may indicate a decrease in at least one selected from the group consisting of liver fat content, AST level, ALT level, and GGT level.

According to one embodiment, the negative prognosis of liver disease or worsening of liver disease may be an increase in one or more selected from the group consisting of liver fat content, AST level, ALT level, and GGT level.

According to one embodiment, if the prognosis of liver disease is negative or the liver disease worsens, the nonalcoholic fatty liver disease may progress to nonalcoholic steatohepatitis, nonalcoholic fatty liver-associated cirrhosis, or liver cancer.

In another aspect, glycine, glyceric acid, serine, threonine, proline, β-alanine from a biological sample isolated from a subject to which a drug candidate is administered β-alanine), phenylalanine, pyroglutamic acid, α-ketoglutaric acid, glutamic acid, citric acid, decanoic acid, mi Myristic acid, palmitoleic acid, palmitic acid, linoleic acid, oleic acid, stearic acid, oleamide, oleic acid One selected from the group consisting of nitrile (Oleanitrile), monopalmitin (Monopalmitin), monostearin (Monostearin), α-hydroxyisobutyric acid, glycerol (Glycerol), and phosphoric acid (Phosphoric acid) measuring the metabolite concentration above; and

It provides a screening method for a drug for preventing, improving, or treating liver disease, comprising comparing the concentration of the metabolite measured in the above step with the concentration of the corresponding metabolite in a control sample.

According to one embodiment, the control sample may be a biological sample isolated from a normal individual without liver disease or a biological sample isolated from a subject before the drug candidate is administered.

According to one embodiment, the biological sample isolated from the subject to which the drug candidate is administered is after 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, and 8 weeks after the drug candidate is administered. , after 10 weeks, after 12 weeks, after 4 months, after 6 months, after 8 months, after 10 months, or after 12 months.

In the present specification, comparing the concentration of a metabolite measured in a subject sample to which a drug candidate is administered with the concentration of a corresponding metabolite in a control sample means comparing the concentration of a metabolite of the same type. For example, it may be comparing the concentration of glycine in the plasma of a subject to which the drug candidate is administered with the concentration of glycine in the plasma of the subject before the drug candidate is administered.

According to one embodiment, the drug candidate is a substance expected to be able to treat liver disease or a substance expected to improve the prognosis of liver disease, for example, a compound, nucleic acid, protein, polypeptide, microbial culture medium. , food, and may be at least one selected from the group consisting of extracts of natural products.

According to one embodiment, the nucleic acid may be one selected from the group consisting of DNA, RNA, antisense nucleic acid, microRNA, siRNA, shRNA, aptamer, LNA (locked nucleic acid), and PNA (peptide nucleic acid). However, the present invention is not limited thereto.

According to one embodiment, the drug candidate may be administered by a method conventionally performed in the industry according to its characteristics, and may be administered orally or parenterally (eg, intravenous administration, intraperitoneal administration, intramuscular administration). , transdermal administration or subcutaneous administration).

According to one embodiment, the administration of the drug candidate may be administered once a day, may be administered in several divided doses, may be administered in an amount that can obtain the maximum effect with a minimum amount without side effects, which It can be readily determined by one of ordinary skill in the art.

According to one embodiment, the biological sample may be blood or plasma.

According to one embodiment, in the method for screening a drug for preventing, ameliorating, or treating liver disease, when the concentration of the metabolite measured in the above step is reduced than the concentration of the corresponding metabolite in the control sample, the drug candidate administered to the subject The substance may be selected as effective as a drug for preventing, ameliorating, or treating liver disease.

According to one embodiment, the screening method of the drug for preventing, improving, or treating liver disease is glycine, glyceric acid, serine, threonine, proline, β -alanine (β-alanine), phenylalanine (Phenylalanine), pyroglutamic acid (Pyroglutamic acid), α-ketoglutaric acid (α-ketoglutaric acid), glutamic acid (Glutamic acid), citric acid (Citric acid), decanoic acid (Decanoic acid) ), Myristic acid, Palmitoleic acid, Palmitic acid, Linoleic acid, Oleic acid, Stearic acid, Oleamide ), olenitrile (Oleanitrile), monopalmitin (Monopalmitin), monostearin (Monostearin), α-hydroxyisobutyric acid (α-hydroxyisobutyric acid), glycerol (Glycerol), and from the group consisting of phosphoric acid (Phosphoric acid) When the concentration of one or more selected metabolites is measurably significantly reduced in a subject sample administered with a drug candidate as compared to a control sample (e.g., 10% or more, 20% or more, 30% or more, 40% or more) , 50% or more, 60% or more, or 70% or more) may be selecting the drug candidate administered to the subject as effective as a drug for preventing, ameliorating, or treating liver disease.

According to one embodiment, the liver disease may be non-alcoholic fatty liver disease (NAFLD).

According to one embodiment, the subject before administration of the drug candidate may be a subject having nonalcoholic fatty liver disease. Non-alcoholic fatty liver disease is the same as described above.

According to one embodiment, before administration of the drug candidate, the level of one or more liver enzymes selected from the group consisting of AST, ALT, and GGT exceeds the upper limit of the normal range, and 3 of the upper limit of the normal range. It may be a mammal with a subfold level.

According to one embodiment, the subject before administration of the drug candidate may be a mammal having one or more characteristics selected from the following characteristics;

(1) an aspartate aminotransferase (AST) level of 40 IU/L or more (or more than 40 IU/L) and less than 120 IU/L;

(2) an alanine aminotransferase (ALT) level greater than or equal to 40 IU/L (or greater than 40 IU/L) and less than 120 IU/L, and

(3) a gamma-glutamyl transferase (GGT) level greater than or equal to 63 IU/L (or greater than 63 IU/L) and less than 189 IU/L if the subject is a male, or greater than or equal to 35 IU/L (or greater than or equal to 35 IU/L) if the subject is a female L) and a GGT level of less than 105 IU/L.

According to one embodiment, the step of measuring the metabolite concentration may be performed using one or more selected from the group consisting of nuclear magnetic resonance spectroscopy (NMR), chromatography, and mass spectrometry. Nuclear magnetic resonance spectroscopy, chromatography, and mass spectrometry are the same as described above.

In another aspect, in a biological sample isolated from a mammal, glycine, glyceric acid, serine, threonine, proline, β-alanine, phenylalanine ( Phenylalanine), Pyroglutamic acid, α-ketoglutaric acid, Glutamic acid, Citric acid, Decanoic acid, Myristic acid, Palmitoleic acid, Palmitic acid, Linoleic acid, Oleic acid, Stearic acid, Oleamide, Oleanitrile, Mono For quantitation of one or more metabolites selected from the group consisting of palmitin, monostearin, α-hydroxyisobutyric acid, glycerol, and phosphoric acid It provides a kit for diagnosing liver disease, including a formulation or a metering device.

According to one embodiment, the mammal may include humans and primates such as monkeys, and rodents such as rats and mice.

As used herein, the term 'quantitative preparation' refers to a preparation for quantitatively detecting the metabolite from a biological sample isolated from a mammal, and the preparation is not particularly limited.

According to one embodiment, the quantification agent interacts with the metabolite to generate a signal (eg, a fluorescent, luminescent or radioactive signal) capable of complementary binding, a primer, a probe, an aptamer, a small molecule compound, a protein , a ligand or an antibody.

According to one embodiment, the kit for diagnosing liver disease may further include a detection reagent. The detection reagent may be a conjugate labeled with a detector such as a chromogenic enzyme, a fluorescent material, a radioactive isotope, or a colloid. The chromogenic enzyme may be peroxidase, alkaline phosphatase, or acid phosphatase, and the fluorescent material is fluorescein carboxylic acid (FCA), fluorescein isothiocyanate (FITC), fluorescein thiourea (FTH), 7- Acetoxycoumarin-3-yl, fluorescein-5-yl, fluorescein-6-yl, 2',7'-dichlorofluorescein-5-yl, 2',7'-dichlorofluorescein- 6-yl, dihydro tetramethylrosamine-4-yl, tetramethylrhodamine-5-yl, tetramethylrhodamine-6-yl, 4,4-difluoro-5,7-dimethyl-4-bora -3a,4a-diaza-s-indacene-3-ethyl, or 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3 -Ethyl, Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate (RITC), PE (Phycoerythrin), or rhodamine.

According to one embodiment, the kit for diagnosing liver disease may additionally include a device capable of detecting a signal generated when an agent for quantification and a metabolite interact. For example, the device capable of detecting a signal may be any one or more selected from the group consisting of an ultraviolet spectrometer, an infrared spectrometer, an ultraviolet-visible light (UV-vis) spectrometer, and a spectrophotofluorometer.

According to one embodiment, the quantitative device is for measuring the concentration of each metabolite, and may be any one or more selected from the group consisting of nuclear magnetic resonance spectroscopy (NMR), chromatography, and mass spectrometry. have.

The method for providing information for diagnosing liver disease according to an embodiment is non-invasive, because it uses a sample isolated from an individual, and can easily diagnose liver disease, and can examine various types of metabolite patterns. The accuracy is excellent.

1 is a schematic diagram showing subject information in the stages of recruitment, assignment, and analysis during the course of an experiment. 1 , placebo indicates a placebo group, Low (low-dose) indicates a low-dose group, and High (high-dose) indicates a high-dose group. The same applies to the drawings below.
2 shows the liver fat content (%) in the placebo group and the pinitol intake group (low-dose group and high-dose group).
3A to 3C show liver enzyme levels measured in the placebo group and the pinitol intake group (low-dose group and high-dose group). Specifically, Figure 3a shows the ALT level (U / L), Figure 3b shows the AST level (U / L), Figure 3c shows the GGT level (U / L). In FIGS. 2 and 3A to 3C , the box represents the 25th to the 75th percentile, the black line represents the median, and the whisker represents the 10th to the 90th percentile.
4A to 4C show the results of measuring triglycerides after ingestion of a high-fat diet and pinitol (or placebo). Specifically, Figure 4a shows the triglyceride concentration (mg / dL) 0, 2, 4 hours after ingestion of a high fat diet and pinitol (or placebo), Figure 4b is AUC (area under the curve; mg * min / dL) values, and FIG. 4c shows pre-peak AUC (mg*min/dL) values.
5a to 5c show the results of measuring postprandial urine MDA after ingestion of a high-fat diet and pinitol (or placebo). Specifically, Figure 5a shows the MDA concentration (μmol / g) 0, 2, 4 hours after ingestion of a high fat diet and pinitol (or placebo), Figure 5b is AUC (area under the curve; μmol * min / g) value , and Figure 5c shows the pre-peak AUC (pre-peak AUC; μmol*min/g) value.
6 shows the results of measuring the levels (μg/mg) of pinitol and myo-inositol in the urine of the high-dose pinitol intake group.
7A and 7B show the results of analysis of plasma metabolites before and after ingestion of pinitol (or before and after improvement of NAFLD symptoms). Specifically, Figure 7a shows metabolites differentiated by PCA and OPLS-DA before and after improvement of NAFLD symptoms. In FIG. 7A , △ indicates metabolites in blood collected from NAFLD subjects before ingestion of pinitol, and △ indicates metabolites in blood collected from subjects whose NAFLD symptoms were improved by ingestion of pinitol for 12 weeks. 7B shows a heat map constructed based on metabolites with VIP>0.7 and P<0.05 values before and after improvement of NAFLD symptoms.

Hereinafter, the present invention will be described in more detail through reference examples and examples. However, these Reference Examples and Examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited to these Reference Examples and Examples.

Reference Example 1. Materials

A capsule containing pinitol powder and dextrin as a color-matched placebo was supplied from Amicogen, Inc. (Jinju, Gyeongsangnam-do, Korea) and used.

Pinitol was extracted from carob pods using water, and pre-cultured Saccharomyces carlsbergensis was inoculated and cultured for 72 hours to increase the pinitol content in the extract. In order to remove the microbial mass generated during the culture, filtration using charcoal column chromatography and ion exchange chromatography, concentrating the resulting fraction, and then adding ethanol to crystallize the extract containing pinitol Pinitol powder was prepared.

The nutritional components of the pinitol powder are listed in Table 1. Pinitol powder contains 1.3% sodium and other ingredients in addition to carbohydrates, proteins and fats.

Reference example 2. Subject recruitment and pinitol Intake

Subjects were recruited from Bundang Cha Hospital (Bundang, Gyeonggi, Korea) for a randomized, double-blind, placebo-controlled trial. (1) liver enzymes (at least one of ALT, AST and GGT) are above the upper limit of normal (AST: 40 IU/L or higher, ALT: 40 IU/L or higher, and GGT: 63 IU/L or higher in men, or in women) 35 IU/L or more), not exceeding 3 times the upper limit of normal (AST: less than 120 IU/L, ALT: less than 120 IU/L, and GGT: less than 189 IU/L for men or less than 105 IU/L for women), ( 2) Non-alcoholic fatty liver disease (hereinafter, NAFLD) was confirmed through ultrasound examination (3) 177 NAFLD subjects 20 years of age or older were recruited.

Subjects were excluded if they met the following criteria and a total of 87 subjects were excluded: (1) taking medications and/or dietary supplements that could affect liver function; (2) recommended Food Score (RFS) > 36; (3) smoking ≥ 1 pack/day; (4) Drinking intake: Women>140g/week, Men>210g/week; (5) continuous consumption of soy or soy products (tofu, soy milk, etc.) for 2 weeks prior to the first visit; (6) Presence or history of disease (viral liver disease, severe liver disease, primary biliary cirrhosis, liver disease induced by drugs or surgery, hereditary liver disease, infectious disease, severe cardiovascular disease, digestion organ disease, kidney disease, thyroid disease, autoimmune disease, malignancy, multisystem failure, Inflammatory Bowel Disease, severe mental illness, HIV positive, etc.); (7) organ transplant or bone marrow transplant; (8) expected use of a prohibited substance; or (9) pregnancy or lactation.

After obtaining written consent, the experiment was conducted on 90 eligible subjects who met the above conditions. Randomization was performed using computer-generated random numbers, and group assignments were blinded for both raters and participants. Subjects were randomly assigned to one of the following three groups. A composition containing no pinitol component and containing dextrin having a nutritional component similar to pinitol was used as a placebo.

(1) Placebo group: Take 2 capsules (about 300 mg) containing dextrin (98.0%, about 294 mg) and magnesium stearate (2.0%, about 6 mg) with water once a day for 12 weeks.

(2) Low-dose finitol intake group (hereinafter, low-dose group): 2 capsules (about 300 mg) containing finitol powder (52.6%, about 157.8 mg), dextrin (45.4%), and magnesium stearate (2.0%) Take with water once a day for 12 weeks.

(3) High-dose finitol intake group (hereafter, high-dose group): 2 capsules (about 300 mg) containing finitol powder (87.7%, about 263.1 mg), dextrin (10.3%), and magnesium stearate (2.0%) Take with water once a day for 12 weeks.

Fasting blood samples were collected at baseline (week 0) and week 12. A fasting blood sample was taken from a vein in the arm. Venous blood samples collected at week 0 and collected at week 12 using K2-EDTA tubes (BD Biosciences, San Jose, CA, USA) and serum separation tubes (SST tubes; BD Biosciences, San Jose, CA, USA), respectively. Plasma and serum were isolated from the collected venous blood samples. The separated plasma and serum were placed in Eppendorf tubes and frozen at -80°C until analysis.

For the postprandial test, within 15 minutes after a fasting blood sample was taken at week 12, the subject was subjected to a capsule or dextrin containing pinitol in the above composition along with a high fat/high glucose formula. ingested capsules containing High Fat/High Glucose (900 kcal total): 58.9% energy from fat, 33.3% from carbohydrates, and 7.6% energy from protein) consists of palm oil (60 g), dextrose (83.5 g), protein powder (20 g) and water became Postprandial blood samples were collected 0, 2 and 4 hours after ingestion of high fat/high glucose and test substance (pinitol or placebo). The postprandial blood sample was performed in the same manner as the fasting blood sample collection method described above.

Reference Example 3. Magnetic Resonance Imaging (MRI)

MRI was performed using a 1.5T MRI system (Optima MR 360, GE Healthcare, Milwaukee, IL, USA) with a 12-channel phased-array torso coil, and the liver fat content (liver fat content) was measured. The MRI-determined proton density fat fraction (PDF) estimate was calculated by applying the confounder-corrected chemical shift-encoded MRI (CSE-MRI) method. To generate a confounder-corrected PDFF map across the liver, quantitative CSE-MR images were acquired using a multiecho 3D spoiled gradient-echo sequence. Acquisition parameters typically include: repetition time (TR), 13.6 ms; 6 echoes acquired in one TR (initial echo time [TE], 1.2 ms; ΔTE, 2.0 ms); a field of view of 44Х44 cm; 256Х160 matrix; 8mm slice thickness; 32 pieces; 5° flip angle (to minimize T1-related bias); and ± 125 kHz receiver bandwidth.

PDFF MR images were adjusted to allow direct placement of ROI regions of interest, yielding estimates of PDFF expressed as absolute percentages (0-100%). Three ROIs (<1 cm) located in the right hepatic lobe were established in each MRI to avoid large vessels and bile ducts, local lesions and obvious image artifacts.

Reference Example 4. Biochemical measurement

Reference Example 4.1 Measurement of biochemical markers

Serum triglyceride (TG), total cholesterol (TC), and low density lipoprotein cholesterol (LDL-C; low density lipoprotein cholesterol) using an automatic analyzer (Cobas c702 analyzer; Hitachi High-Technologies, Osaka, Japan) , high density lipoprotein cholesterol (HDL-C), liver enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT)) were measured.

Reference Example 4.2 Glutathione peroxidase activity measurement

Glutathione peroxidase (GPx) activity was measured using a glutathione peroxidase assay kit (Abcam, Cambridge, UK) according to the manufacturer's instructions. Red blood cells were lysed with 4 times distilled water and centrifuged at 10,000×g for 15 minutes at 4°C. A supernatant was obtained after centrifugation, and the obtained supernatant was diluted 100-fold with a sample buffer. For measurement of GPx activity, 100 μl standard and 50 μl dilution samples were mixed with 40 μl GPx activity assay reagent containing assay buffer, glutathione, NADPH, glutathione reductase. After incubation at room temperature for 15 minutes, 10 μl cumen hydroperoxide solution was added. Thereafter, the GPx activity was determined by measuring the absorbance at 340 nm for 5 minutes at intervals of 5 minutes using a microplate reader to determine the GPx activity.

Reference Example 4.3 Glutathione expression measurement

The level of total glutathione (GSH) was measured using a glutathione assay kit (Cayman, Ann Arbor, MI, USA) according to the manufacturer's instructions. Red blood cells were lysed with 4 times distilled water and centrifuged at 10,000×g for 15 minutes at 4°C. A supernatant was obtained after centrifugation, and the obtained supernatant was mixed with an equal amount of 10% (w/v) metaphosphoric acid (Showa denko KK, Tokyo, Japan) to deproteinize before analysis, and 2,000 at room temperature for 3 minutes. Centrifuged at xg. 200 μl of the supernatant was mixed with 10 μl of 4M triethanolamine (Sigma-Aldrich, St. Louis, MO, USA), vortexed immediately, and then diluted with 30-fold MES buffer.

For the measurement of GSH, 50 μl of the diluted sample and standard solution was added to a 96-well plate, and 150 μl assay cocktail (NADPH, glucose-6-phosphate, glutathione reductase (GR), glucose-6-phosphate dehydrogenase) was added to a 96-well plate for 1 minute at 25°C. , and 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB)). Thereafter, the TGSH level was read by measuring the absorbance at 405 nm at 5-minute intervals for 30 minutes using a microplate reader.

Reference Example 5. MDA (Malondialdehyde) analysis

To determine the level of oxidative damage, high-performance liquid chromatography-fluorescence detection (HPLC-FLD; Shiseido, Tokyo, Japan) using a 5 μm Capcell Pak C18 column (4.6 mm id Х 250 mm; Shiseido) was performed. Urine MDA levels were measured by As described in Reference Example 2, the urine collected from the subjects at baseline (week 0) and week 12, or after high-fat diet and finitol intake, was mixed with 0.44 M phosphoric acid (Duksan Pure Chemicals Co., Ltd., Korea) and 42 mM TBA. (2-thiobarbituric acid; Sigma-Aldrich, St. Louis, MO, USA) solution was deproteinized. The deproteinized samples were heated at 95° C. for 1 hour, cooled on ice, and centrifuged at 2,500×g at 4° C. for 3 minutes. The resulting supernatant was filtered with a 0.45 μm PTFE syringe filter (Woongki Science, Seoul, Korea). Using 50 mM potassium phosphate buffer (pH 6.8) in methanol (8:2, v/v) as a mobile phase, HPLC-FLD analysis was performed at 40° C. at a flow rate of 1.0 ml/min. A standard curve was generated using a 1,1,3,3-tetraethoxypropane solution to adjust the peak of the MDA-TBA adduct. MDA was quantified by measuring the peak area in an HPLC chromatogram calibrated against a defined standard. The detector results were recorded using a data management system (EZChrom quick soft version 3.1).

Reference Example 6. Measurement of urine pinitol and myo-inositol

Pinitol and myo-inositol concentrations were analyzed in urine samples collected from subjects in the high-dose pinitol intake group at weeks 0 and 12 using gas chromatography-mass spectrometry (GC-MS). The urine was freeze-dried to equalize the concentration and extracted with 70% methanol. Dicyclohexyl phthalate was added to the urine sample as an internal standard. 10 μl of the extract was completely dried using a CentriVap concentrator (Labconco, Kansas City, MO, USA) at 70° C., and methoxylated with 70 μl of methoxyamine hydrochloride at 70° C. for 90 minutes. It was reacted with 70 μl of N ,O-bis(trimethylsilyl) trifluoroacetamide (N,O-bis(trimethylsilyl) trifluoroacetamide) at 37° C. for 30 minutes for silylation. GC-MS data were obtained on a GC-2010 Plus instrument (Shimazu, Tokyo, Japan) equipped with a DB-5 column (30 m length Х 0.25 mm id, 0.25 μm film thickness; J&W Scientific, Santa Clara, CA, USA). Helium was used as a carrier gas at a constant flow of 1 ml/min.

The oven temperature was held at 70° C. for 2 minutes, increased to 210° C. at a rate of 7° C./min, and then held at 320° C. for 7 minutes. The injector and ion source temperatures were 200 and 230°C, respectively. GC column eluates were analyzed using electron impact ionization mode on a GCMS-TQ 8030 apparatus (Shimadzu, Kyoto, Japan). Electron ionization was performed at 70 eV, and mass data were collected through a full scan in the m/z range of 45-800.

Reference Example 7. Endogenous metabolite analysis in plasma

Plasma separated from the fasting blood samples (week 0 and week 12) of the high-dose pinitol intake group collected as in the method described in Reference Example 2 above using a shaker and a sonicator at room temperature for 10 minutes mixed with methanol. After incubation at 4° C. for 1 hour to precipitate the protein, the mixture was centrifuged at 4° C. at 16,600×g for 10 minutes. The supernatant obtained after centrifugation was filtered using a 0.2 μm polytetrafluoroethylene (PTEE) filter and completely dried using a speed vacuum concentrator. Derivatization was carried out in two steps, oximation and silylation, to volatilize metabolites. The dried sample was oximated with methoxy amine hydrochloride in pyridine at 30° C. for 90 minutes, and silylated with N-methyl-N-trimethylsilyl trifluoroacetamide at 37° C. for 30 minutes to derivatize ( derivatized).

GC-GC was performed using an Agilent 7890 gas chromatograph system (Agilent Technologies, Palo Alto, CA, USA) coupled with an Agilent 7893 autosampler and equipped with a Pegasus HT TOF-MS system (Leco, St. Joseph, MI, USA). TOF-MS analysis was performed. An Rtx-5MS column (length 29.8 m, id 0.25 mm, particle size 0.25 μm; Restek Corp., Bellefonte, PA, USA) was used with helium as the carrier gas at a constant flow rate of 1.5 ml/min. Samples were injected in 1 μl aliquots. The oven temperature was held at 75° C. for 2 minutes, raised to 300° C. at a rate of 15° C./min, and held for 3 minutes. The temperatures of the front inlet, transfer line, and ion source were 250°C, 240°C, and 230°C, respectively. Electron ionization was performed at -70 eV, and mass data were collected through full scanning in the range of 50-100 m/z.

Reference Example 8. Statistical Analysis

All statistical analyzes were performed using the SAS program. The metabolite data obtained in Reference Example 7 were subjected to multivariate statistical analysis using SIMCA software version 14.1 (Umetrics, Inc., Umeε, Sweden). The plasma metabolite analysis results were visualized using principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). Metabolites with variable importance plots (VIP) values >0.7 and P<0.05 were selected as metabolites to provide information for diagnosing liver disease. Heat maps were generated based on selected metabolites using MetaboAnalyst 4.0 (http://metaboanalyst.ca).

Example 1. Subject Characteristics

As in Reference Example 2, a total of 90 eligible subjects were randomly assigned to three groups (placebo group/low-dose group/high-dose group), and 76 subjects (placebo group: 28, low-dose group: 21, high-dose group) : 27) completed the endpoint assessment ( FIG. 1 ).

The characteristics of all subjects at baseline are summarized in Table 2. All subjects were confirmed to have fatty liver by ultrasound. Except for the liver fat content (P = 0.047), there was no significant difference in characteristics between groups at baseline. Liver fat content was lower in the high-dose group, and this variable was considered a covariate in the analysis. The subjects had, on average, ALT of 62.8 ± 3.5 U/L, AST of 37.4 ± 1.6 U/L, and GGT of 69.4 ± 4.4 U/L, and had fatty liver.

Example 2. For liver function pinitol effect of intake

Pinitol restores GOT, GPT, and γ-glutamyl transpeptidase (γ-GTP) to almost normal levels when taken by patients with fatty liver (Korean Patent Publication No. 10-2004-0084168), and is known to be effective in treating fatty liver disease. And it was confirmed that pinitol directly exhibits a therapeutic effect on nonalcoholic fatty liver disease by the following examples.

Example 2.1 Measurement of liver enzymes and liver fat content

To determine the effect of pinitol intake on liver function, liver enzyme (ALT, AST, GGT) levels and liver fat content were evaluated. At baseline (week 0) and week 12 in each group, MRI was performed by the method of Reference Example 3 to measure liver fat content, and liver enzyme levels were measured by the method of Reference Example 4.1, and the results are shown in FIGS. 2 and 3a to It is shown in Figure 3c. In the drawings and tables, Placebo denotes a placebo group, Low denotes a low-dose group, and High denotes a high-dose group.

As shown in FIG. 2 , unlike the placebo group, the liver fat content decreased over time in the pinitol intake group (low-dose group and high-dose group). In particular, the liver fat content at week 12 compared to baseline in the low-dose group was significantly ( P = 0.010) decreased. Even in the high-dose group, the liver fat content decreased at week 12 compared to the baseline, but the liver fat content at week 0 was significantly lower than that of other groups, indicating that the amount of decrease was smaller than in the low-dose group.

As shown in FIGS. 3A to 3C , the ALT level significantly decreased at week 12 in the pinitol intake group (low-dose group and high-dose group), and the AST level decreased at week 12 in the pinitol intake group, especially in the low-dose group. Significantly decreased. GGT levels were significantly decreased at week 12 in the high-dose group.

Levels of liver enzymes may increase due to liver damage such as damage to hepatocellular membranes, and since liver fat content and liver enzyme levels increase in NAFLD patients, a decrease in liver fat content and liver enzyme levels following pinitol intake may improve NAFLD symptoms can be considered as

Example 2.2 Blood Lipid Profile and Oxidative Stress

The values for the lipid profile and oxidative stress-related markers upon ingestion of pinitol were measured in the same manner as in Reference Examples 4 and 5, and the results are shown in Table 3.

As shown in Table 3, the levels of total cholesterol and low-density lipoprotein cholesterol were decreased in the pinitol intake group (high-dose group and low-dose group), unlike the placebo group.

In addition, to evaluate oxidative damage, urine MDA was measured to determine the degree of lipid peroxidation. As shown in Table 3, urine MDA increased significantly at week 12 compared to baseline in the placebo group, but remained unchanged (low-dose group) or decreased (high-dose group) in the pinitol intake group. From this, it was found that the pinitol intake group had stronger resistance to oxidative stress than the placebo group.

As shown in Table 3, the GPx concentration was significantly increased in the finitol intake group and significantly decreased in the placebo group after 12 weeks of finitol intake. In addition, it was confirmed that the GSH concentration also significantly increased in the pinitol intake group.

Example 2.3 postprandial oral lipid tolerance test

Example 2.3.1 Postprandial triglyceride following ingestion of pinitol

As described in Reference Example 2 above, postprandial blood samples were collected at 0, 2 (120 minutes), and 4 hours (240 minutes) after ingestion of pinitol or placebo with high-fat diet intake at week 12, and in Reference Example 4.1 The serum triglyceride (TG) concentration was measured as in the method, and the results are shown in FIGS. 4A to 4C . Figure 4a shows the triglyceride concentration (mg / dL) 0, 2, 4 hours after ingestion of a high fat diet and pinitol (or placebo), Figure 4b is AUC (area under the curve; area under the curve; mg * min / dL ) values, and FIG. 4c shows pre-peak AUC (mg*min/dL) values.

As shown in Figures 4a to 4c, the experimental group ingesting pinitol with a high-fat diet inhibited the increase in serum TG when compared to the placebo group. Postprandial TG levels were changed with time (P < 0.0001), and there was a concentration-dependent inhibitory effect on serum TG increase in the pinitol intake group. In particular, in the high-dose group, postprandial TG levels remained the lowest at all time points (0, 2, and 4 hours).

Example 2.3.2 Postprandial urine MDA according to pinitol intake

As described in Reference Example 2 above, postprandial urine samples were collected at 0, 2 (120 minutes), and 4 hours (240 minutes) after ingestion of pinitol or placebo with high-fat diet intake at week 12, and in Reference Example 5 The MDA concentration was measured as in the method, and the results are shown in FIGS. 5A to 5C. Figure 5a shows the MDA concentration (μmol/g) after 0, 2, 4 hours after ingestion of a high-fat diet and pinitol (or placebo), and Figure 5b is AUC (area under the curve; μmol*min/g) values, and FIG. 5c shows pre-peak AUC (μmol*min/g) values.

As shown in FIGS. 5A to 5C , the experimental group ingesting pinitol with a high-fat diet significantly inhibited the increase in urine MDA when compared to the placebo group.

Through the results of Examples 2.3.1 and 2.3.2, pinitol inhibits the rise of serum triglycerides and urine MDA due to high fat intake, thereby improving liver function and treating liver disease, and can also prevent liver disease. confirmed that .

Example 3. Confirmation of ingestion of pinitol

In order to evaluate the exposure, as in the method of Reference Example 6, urine was collected from the high-dose pinitol intake group at weeks 0 and 12, and the levels of pinitol and myo-inositol were measured and shown in FIG. 6 .

As shown in FIG. 6 , urine pinitol was not found at baseline (week 0) and was found after 12 weeks of pinitol intake. There was no change in the concentration of urine myo-inositol between weeks 0 and 12.

Example 4. Plasma Metabolite Analysis

To select biomarkers for liver disease diagnosis, metabolites were analyzed by performing GC-TOF-MS on plasma samples collected before and after NAFLD symptoms were improved by ingestion of pinitol as in the method of Reference Example 7 above. In order to investigate the change in metabolite pattern according to whether or not NAFLD symptoms improved, multivariate analysis was performed. -discriminant analysis; metabolites clearly distinguished by orthogonal partial least-squares discrimination analysis) are shown in FIG. 7a. In FIG. 7A , △ indicates metabolites in blood collected from NAFLD subjects before ingestion of pinitol, and △ indicates metabolites in blood collected from subjects whose NAFLD symptoms were improved by ingestion of pinitol for 12 weeks. A heat map constructed based on significantly different metabolites is shown in FIG. 7B . 25 metabolites with VIP>0.7 and P<0.05 values were selected in order to select the major metabolites that were significantly different before and after the improvement of NAFLD symptoms, and are shown in Table 4 below. The levels of 25 metabolites listed in Table 4 below were significantly reduced in subjects whose NAFLD symptoms were improved or treated with pinitol intake.

The metabolites selected above were selected as biomarkers for diagnosing liver disease by metabolites whose levels decreased, not increased according to ingestion of pinitol, in subjects with improved NAFLD symptoms, which were related to pinitol or pinitol. It is distinct from the result of the body's reaction.

metabolite P-value Decrease (%) One Glycine <.0001 32.0 2 Glyceric acid 0.0161 25.0 3 Serine 0.0031 18.4 4 Threonine 0.0426 16.2 5 Proline <.0001 59.4 6 β-alanine 0.0363 9.2 7 Phenylalanine 0.0155 19.2 8 Pyroglutamic acid <.0001 30.5 9 α-ketoglutaric acid
(α-ketoglutaric acid) 0.0083 45.8 10 Glutamic acid 0.0003 53.3 11 Citric acid 0.0179 12.3 12 Decanoic acid <.0001 72.2 13 Myristic acid 0.0006 34.3 14 Palmitoleic acid 0.0017 49.5 15 Palmitic acid <.0001 35.3 16 Linoleic acid <.0001 29.7 17 Oleic acid <.0001 44.5 18 Stearic acid <.0001 35.1 19 Oleamide <.0001 19.0 20 Oleanitrile 0.0145 44.0 21 Monopalmitin 0.0009 24.9 22 Monostearin <.0001 22.9 23 α-hydroxyisobutyric acid
(α-hydroxyisobutyric acid) 0.0060 45.2 24 Glycerol 0.0158 28.1 25 Phosphoric acid 0.0392 16.2

Claims (14)

delete delete delete delete delete delete Metabolites of Decanoic acid, Myristic acid, Palmitic acid, and Stearic acid in blood or plasma isolated from a subject to which a drug candidate is administered measuring the concentration; and
A drug for preventing, improving, or treating non-alcoholic fatty liver disease (NAFLD), comprising comparing the concentration of the metabolite measured in the above step with the concentration of the corresponding metabolite in the control sample screening method. According to claim 7, wherein the step of measuring the metabolite concentration in the blood or plasma, pyroglutamic acid (Pyroglutamic acid), glycine (Glycine), glyceric acid (Glyceric acid), serine (Serine), threonine (Threonine), Proline, β-alanine, Phenylalanine, α-ketoglutaric acid, Glutamic acid, Citric acid, Palmitoleic acid ), Linoleic acid, Oleic acid, Oleamide, Oleanitrile, Monopalmitin, Monostearin, α-hydroxyisobutyric acid (α- hydroxyisobutyric acid), glycerol (Glycerol), and phosphoric acid (Phosphoric acid), the screening method further comprising measuring the concentration of one or more metabolites selected from the group consisting of. The method of claim 7 or 8, wherein when the concentration of the metabolite measured in the step is reduced than the concentration of the corresponding metabolite in the control sample, the drug candidate administered to the subject is used for preventing, improving, or treating liver disease. A screening method that is selected as effective as a drug for use. The method of claim 7 , wherein the subject is a mammal. The screening method according to claim 7, wherein the subject before administration of the drug candidate is a subject having non-alcoholic fatty liver disease (NAFLD). The screening method according to claim 7, wherein the subject before administration of the drug candidate is a mammal having one or more characteristics selected from the following characteristics;
(1) AST (aspartate aminotransferase) level of 40 IU/L or more and less than 120 IU/L,
(2) an alanine aminotransferase (ALT) level of 40 IU/L or more and less than 120 IU/L, and
(3) a gamma-glutamyl transferase (GGT) level of 63 IU/L or more and less than 189 IU/L when the subject is a male, or a GGT level of 35 IU/L or more and less than 105 IU/L when the subject is a female. The method of claim 7, wherein the step of measuring the metabolite concentration is performed using one or more selected from the group consisting of nuclear magnetic resonance spectroscopy (NMR), chromatography, and mass spectrometry. screening method. delete

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