Detection of liver diseases

Abstract

The present invention relates to a method for detecting, staging, monitoring, or predicting the prognosis of non-alcoholic steatohepatitis (NASH) in a subject. The method includes a step of measuring the concentration of an exogenous substrate of an enzyme and / or a step of measuring the concentration of a metabolite of the substrate in a biological sample obtained from the subject. The substrate is a generally recognized as safe (GRAS) compound.

Description

Technical Field

[0001] The present invention relates to methods for the detection of non-alcoholic steatohepatitis (NASH), as well as related methods and kits.

Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) encompasses the entire histological spectrum ranging from simple benign hepatic steatosis to non-alcoholic steatohepatitis (NASH) characterized by lipid accumulation, inflammation, ballooning of hepatocytes, and varying degrees of fibrosis. NASH can progress to cirrhosis or hepatocellular carcinoma (HCC). NASH, unlike non-alcoholic fatty liver (NAFL), has a very high likelihood of progressing to cirrhosis, liver failure, and liver cancer. The prevalence of NAFLD is increasing, which is associated with the increasing number of obesity cases. Despite the growing recognition of obesity-related liver diseases, the etiology of NAFLD and NASH remains poorly understood.

[0003] Based on the prevalence of NAFLD, NASH-induced cirrhosis is expected to become the most common indication for liver transplantation in the future. Distinguishing NASH from simple steatosis is important for the clinical management of NAFLD patients and for reducing mortality (Chen et al., Radiology. 2011 Jun; 259(3): 749-756).

[0004] The only test approved for NASH diagnosis is liver biopsy, which is an invasive procedure that can cause complications. Alternative methods are not sufficient in performance in the early stages of NASH, and overall, this limits the early detection of NASH and makes it difficult to evaluate the effectiveness of experimental drugs. Therefore, there is a need for alternative diagnostic tests, especially tests that can diagnose NASH and distinguish it from other stages of NAFLD, namely NAFL. Non-invasively identifying both NAFLD and NASH would help significantly reduce the risks associated with the diagnosis of these disease states. Distinguishing NASH from NAFL would enable earlier lifestyle modifications, medical interventions, cancer screening, and overall improvement in outcomes.

[0005] The use of exogenous volatile organic compound (EVOC®) probes for detecting liver diseases by monitoring the metabolism processing of exogenous compounds by monitoring induced volatolomics - breath is described in WO2019220145. The use of the probes described herein for the detection, diagnosis, staging, monitoring, and prognosis prediction of NASH provides an alternative test for NASH.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Summary of the Invention

Problems to be Solved by the Invention

[0008] The present inventors have shown that NASH-induced metabolic changes can be detected using EVOC probes, which are safe for human ingestion and appear in exhaled breath along with their bioproducts after administration. These data demonstrate that breath analysis using EVOC probes for induced volatolomics can be used for NASH detection tests, as well as for screening, diagnosis, staging, monitoring, and prognosis prediction.

[0009] In this approach, one or more target compounds that appear in the exhaled breath of a subject and are metabolized by the liver are measured for the exhaled breath after administration. The amount of the compound in the subject's exhaled breath depends on the efficiency of the liver in removing the compound after administration. Therefore, the amount thereof in the exhaled breath represents liver function. To enhance diagnostic accuracy, volatile bioproducts can alternatively or additionally be detected. Therefore, the amount of the compound in the exhaled breath can be used as a surrogate for liver function affected in NASH.

Means for Solving the Problems

[0010] Therefore, the present inventors have developed a non-invasive test for detecting and staging non-alcoholic fatty liver disease (NAFLD) that has progressed to the stage of non-alcoholic steatohepatitis (NASH) in a subject having or suspected of having a liver disease with respect to detection, diagnosis, staging, monitoring, and prognosis prediction (i.e., at risk of progression to a more advanced liver disease stage).

[0011] Accordingly, in a first aspect, the present invention is a method for detecting or predicting the prognosis of NASH in a subject, comprising the step of measuring the concentration of an exogenous substrate of an enzyme in a biological sample obtained from the subject, and / or the step of measuring the concentration of a metabolite of the substrate, wherein the substrate is a generally recognized as safe (GRAS) compound, and the enzyme is an aldo-keto reductase (AKR) or an alcohol dehydrogenase or a cytochrome P450 (CYP) or an aldehyde dehydrogenase or a glycine N-acyltransferase, related to the method.

[0012] The method further comprises the step of determining the stage of NASH, and the stage of NASH is selected from NASH without fibrosis, NASH with fibrosis, NASH with hepatocellular carcinoma (HCC), or NASH with cirrhosis such as decompensated cirrhosis.

[0013] In one embodiment, the enzyme is aldo-keto reductase (AKR).

[0014] In one embodiment, AKR is an AKR family 1 member.

[0015] In one embodiment, AKR is AKR1B10. In one embodiment, the substrate is selected from aldehydes and / or alcohols.

[0016] In one embodiment, the substrate is nonanal, and / or the metabolite is nonanol.

[0017] In one embodiment, the substrate is 1-nonanal, and / or the metabolite is 1-nonanol.

[0018] In one embodiment, the substrate is trans-2-hexenal, and / or the metabolite is trans-2-hexanol.

[0019] In one embodiment, the substrate is hexanal, and / or the metabolite is hexanol.

[0020] In one embodiment, the substrate is benzaldehyde and / or the metabolite is benzyl alcohol.

[0021] In one embodiment, the substrate is citral and / or the metabolite is nerol.

[0022] In one embodiment, the enzyme is alcohol dehydrogenase.

[0023] In one embodiment, the substrate is butanol and / or the metabolite is butanone.

[0024] In one embodiment, the substrate is 2-butanol and / or the metabolite is 2-butanone.

[0025] In one embodiment, the substrate is 2-pentanone.

[0026] In one embodiment, the substrate is 2-pentanone and / or the metabolite is 2-pentanol, 3-hydroxy-2-pentanone or 2,3-pentanediol.

[0027] In one embodiment, the substrate is benzyl alcohol and the metabolite is benzaldehyde and / or benzoic acid.

[0028] In one embodiment, the enzyme is aldehyde dehydrogenase.

[0029] In one embodiment, the substrate is benzaldehyde and the metabolite is benzoic acid.

[0030] In one embodiment, the enzyme is cytochrome P450 (CYP).

[0031] In one embodiment, the CYP is CYP1A1, CYP1A2, CYP1B1, CYP2, CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP2W1, CYP3, CYP3A4, CYP3A5, CYP3A7 or CYP3A43. In one embodiment, the enzyme is CYP2C19, CYP2C9 and / or CYP3A4.

[0032] In one embodiment, the substrate is 2-butanone and the metabolite is 3-hydroxy-2-butanone and / or 2,3-butanediol.

[0033] In one embodiment, the substrate is 2-pentanone and the metabolite is 2,3-pentanediol.

[0034] In one embodiment, the enzyme is glycine N-acyltransferase.

[0035] In one embodiment, the substrate is benzoic acid and the metabolite is hippuric acid.

[0036] In one embodiment, the substrate is labeled with, for example, 12C, 13C, 14C, 2H, 14N or 18O.

[0037] In one embodiment, the substrate is unlabeled.

[0038] In one embodiment, the biological sample is selected from exhaled breath, urine, blood, serum, and / or tissue.

[0039] In one embodiment, the method includes establishing a value of the test subject based on the concentration of the substrate or metabolite in the test subject.

[0040] In one embodiment, the value of the test subject is compared with one or more reference values, and the possibility of NASH is indicated by the difference between the value of the test subject and the reference value.

[0041] In one embodiment, the reference value is the value of a subject diagnosed with NASH.

[0042] In one embodiment, the reference value is the value of a subject diagnosed with non-alcoholic fatty liver disease (NAFLD).

[0043] In one embodiment, the reference value is the value of a subject having NASH that has progressed to decompensated cirrhosis and / or HCC.

[0044] In one embodiment, the reference value is the value of a healthy subject.

[0045] In one embodiment, the concentrations of two or more exogenous substrates of the enzyme and / or the concentrations of two or more metabolites are measured.

[0046] In one embodiment, the subject has been administered an exogenous substrate of the enzyme.

[0047] In one embodiment, the concentration of the metabolite is measured.

[0048] In another aspect, the present invention is a method for determining the effectiveness of treatment, comprising, in a subject diagnosed with NASH, evaluating the activity of an enzyme by measuring the concentration of an exogenous substrate of the enzyme and / or the concentration of a metabolite of the substrate in a biological sample obtained from the subject, wherein the subject is undergoing treatment for NASH and the enzyme is aldo-ketoreductase (AKR) or alcohol dehydrogenase or cytochrome P450 (CYP).

[0049] In one embodiment, the method includes analyzing a first biological sample obtained from the subject at a first time point and subsequently analyzing one or more additional biological samples obtained from the subject at one or more additional time points or the ratios thereof.

[0050] In one embodiment, the treatment for NASH is a gastric bypass surgery and / or a drug-based treatment including administration of at least one drug.

[0051] In one embodiment, the treatment of NASH is a drug-based treatment including gastric bypass surgery and / or administration of at least one drug selected from statins, incretin mimetics, metformin, rimonabant, thiazolidinedione, and orlistat.

[0052] In another aspect, the present invention relates to a method for monitoring the progression or regression of NASH in a subject, the method including measuring the concentration of an exogenous substrate of an enzyme and / or the concentration of a metabolite of the substrate in a biological sample obtained from the subject, wherein the substrate is a generally recognized as safe (GRAS) compound and the enzyme is aldo-ketoreductase (AKR), alcohol dehydrogenase, or cytochrome P450 (CYP).

[0053] In another aspect, the present invention relates to a kit for detecting or predicting the prognosis of NASH, including a substrate of an enzyme and / or a metabolite of the substrate and a device for capturing a biological sample from a patient.

[0054] In one embodiment, the substrate and / or metabolite is selected from nonanal, butanol, trans-2-hexenal, hexanal, benzaldehyde, citral, nonanol, butanone, trans-2-hexenol, hexanol, benzyl alcohol, nerol, 3-hydroxy-2-butanone, 2,3-butanediol, benzoic acid, hippuric acid, 2-pentanone, and 2,3-pentanediol.

[0055] In another aspect, the present invention relates to the use of an exogenous substrate and / or metabolite of an enzyme whose activity or expression is upregulated or downregulated in NASH, in a method for detecting or predicting the prognosis of NASH, wherein the substrate is selected from nonanal, butanol, trans-2-hexenal, hexanal, citral, benzoic acid, butanone, and 2-pentanone, and the metabolite is selected from nonanol, butanone, trans-2-hexenol, hexanol, benzyl alcohol, nerol, 2-pentanone, 3-hydroxy-2-butanone, 2,3-butanediol, hippuric acid, and 2,3-pentanediol.

[0056] In another aspect, the present invention relates to the use for an in vivo method of detecting or predicting the prognosis of NASH in a subject, the method comprising measuring the concentration of nonanal, butanol, trans-2-hexenal, hexanal, benzaldehyde, citral, 2-pentanone, nonanol, butanone, trans-2-hexenol, hexanol, benzyl alcohol, nerol, 2-pentanol, benzoic acid, hippuric acid, 2,3-butanediol, 3-hydroxy-2-pentanone and / or 2,3-pentanediol in a biological sample obtained from the subject.

[0057] In another aspect, the present invention relates to the use of nonanal, butanol, trans-2-hexenal, hexanal, benzaldehyde, citral, 2-pentanone, nonanol, butanone, trans-2-hexenol, hexanol, benzyl alcohol, nerol, 2-pentanol, benzoic acid, hippuric acid, 2,3-butanediol, 3-hydroxy-2-pentanone and / or 2,3-pentanediol as biomarkers for the detection, staging (i.e., assessing the stage of NASH), diagnosis, monitoring and progression of NASH.

[0058] In another aspect, the present invention relates to a method for distinguishing NASH from other stages of NAFLD in a subject, comprising measuring the concentration of an exogenous substrate of an enzyme and / or the concentration of a metabolite of said substrate in a biological sample obtained from the subject, wherein the substrate is a generally recognized as safe (GRAS) compound.

[0059] In one embodiment, the activity or expression of said enzyme is upregulated or downregulated in NASH.

[0060] In one embodiment, the enzyme is an aldo-ketoreductase (AKR) or an alcohol dehydrogenase or a cytochrome P450 (CYP).

[0061] In another aspect, the present invention relates to a method for detecting or predicting the prognosis of early non-alcoholic steatohepatitis (NASH) in a subject, comprising measuring the concentration of an exogenous substrate of an enzyme and / or the concentration of a metabolite of said substrate in a biological sample obtained from the subject, wherein the substrate is a generally recognized as safe (GRAS) compound. In one embodiment, the activity or expression of said enzyme is upregulated or downregulated in NASH. In one embodiment, the enzyme is an aldo-ketoreductase (AKR) or an alcohol dehydrogenase or a cytochrome P450 (CYP).

[0062] In another aspect, the present invention is a method for determining the stage of NASH in a subject, comprising the step of measuring the concentration of an exogenous substrate of an enzyme in a biological sample obtained from the subject, and / or the step of measuring the concentration of a metabolite of the substrate, wherein the substrate is a generally recognized as safe (GRAS) compound. In one embodiment, the activity or expression of the enzyme is upregulated or downregulated in NASH.

[0063] In one embodiment, the enzyme is an aldo-ketoreductase (AKR), an alcohol dehydrogenase, or a cytochrome P450 (CYP).

[0064] In another aspect of the present invention, the present invention is a method for determining the stage of NASH in a subject, comprising the step of measuring the concentration of an exogenous substrate of an enzyme in a biological sample obtained from the subject, and / or the step of measuring the concentration of a metabolite of the substrate, wherein the substrate is a generally recognized as safe (GRAS) compound.

[0065] In one embodiment, the activity or expression of the enzyme is upregulated or downregulated in NASH.

[0066] In one embodiment, the enzyme is an aldo-ketoreductase (AKR), an alcohol dehydrogenase, or a cytochrome P450 (CYP).

[0067] Accordingly, in another aspect, the present invention is a method for detecting or predicting the prognosis of NASH in a subject, for detecting or predicting the prognosis of early NASH in a subject, for determining the stage of NASH in a subject, for monitoring the progression or regression of NASH in a subject, or for distinguishing NASH from other stages of NAFLD in a subject, comprising the step of measuring the concentration of an exogenous substrate of an enzyme in a biological sample obtained from the subject, and / or the step of measuring the concentration of a metabolite of the substrate, wherein the substrate is a generally recognized as safe (GRAS) compound and the enzyme is not a CYP enzyme.

[0068] In another aspect, the present invention relates to a method for detecting NASH or predicting the prognosis in a subject, for detecting or predicting early NASH in a subject, for determining the stage of NASH in a subject, for monitoring the progression or regression of NASH in a subject, or for differentiating NASH from other stages of NAFLD in a subject, the method comprising measuring the concentration of an exogenous substrate of an enzyme and / or measuring the concentration of a metabolite of the substrate in a biological sample obtained from the subject, wherein the substrate is a generally recognized as safe (GRAS) compound and the substrate is not limonene.

[0069] In another aspect, the present invention relates to a method for determining the effectiveness of treatment, the method comprising, in a subject diagnosed with NASH, evaluating the activity of an enzyme by measuring the concentration of an exogenous substrate of the enzyme and / or measuring the concentration of a metabolite of the substrate in a biological sample obtained from the subject, wherein the enzyme is not a CYP enzyme or the substrate is not limonene.

[0070] In all of the above aspects, the combination of substrate and / or metabolite can be used as further described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0071]

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DETAILED DESCRIPTION OF THE INVENTION

[0072] The present invention will be further described below. In the following passages, various aspects of the present invention are defined in more detail. Each aspect so defined can be combined with any other aspect unless clearly indicated otherwise. In particular, any feature indicated as being preferred or advantageous can be combined with any one or more other features indicated as being preferred or advantageous.

[0073] Method The present invention provides a method for detecting, staging, screening, diagnosing, monitoring or predicting the prognosis of NAFLD that has progressed to the stage of NASH. Advantageously, the inventors have developed a method for detecting NASH, which is performed on a biological sample and can be non-invasive or minimally invasive. Furthermore, the inventors have also developed a method for identifying the stage of the disease, i.e., how far the disease has progressed, and distinguishing NASH from NAFL.

[0074] In a first aspect, the present invention is a method for detecting or predicting the prognosis of NAFLD that has progressed to the stage of NASH in a subject, comprising the step of measuring the concentration of an exogenous substrate of a NASH-specific enzyme and / or the concentration of a metabolite of the substrate in a biological sample obtained from the subject, wherein the substrate is a generally recognized as safe (GRAS) compound and the enzyme is an aldo-keto reductase (AKR) or alcohol dehydrogenase or cytochrome P450 (CYP) or aldehyde dehydrogenase or glycine N-acyltransferase.

[0075] NAFLD is a term that refers to a spectrum of conditions caused by the accumulation of fat in the liver. This term encompasses a disease spectrum that includes mild, benign forms of the disease known as fatty liver or NAFL, where there is an accumulation of fat in the liver. This disease state can progress to a more severe form known as NASH, where the liver becomes inflamed, and this stage may be referred to in this specification as NASH without fibrosis. NASH can subsequently progress to NASH with fibrosis, where scar tissue forms around the liver and adjacent blood vessels due to persistent inflammation. The most severe form of this disease is NASH with cirrhosis, which can occur after long-term inflammation and result in liver atrophy and scarring. NASH with cirrhosis causes permanent damage to the liver and can lead to the development of liver failure and liver cancer (hepatocellular carcinoma (HCC)).

[0076] The severity of NAFLD can be described in three stages. Stage 1 is characterized by simple fatty liver (i.e., NAFL or hepatic steatosis). Fat begins to accumulate in individual cells, but liver function is normal. Usually, there are no symptoms, and patients may not be aware that they have this disease state. The fat deposits are considered harmless, but it is important to prevent the disease from progressing to the next stage.

[0077] Stage 2 is often referred to as NASH. NASH is a more active form of this disease state where the liver becomes inflamed. Inflammation is the body's healing response to injury or damage, and in this case, it is a sign that hepatocytes have been damaged. People with NASH may experience dull or throbbing pain in the upper right abdomen (below the right rib cage). NASH may or may not be accompanied by fibrosis.

[0078] Stage 3 is often characterized by cirrhosis. In this most severe stage, bands of scar tissue and nodules of hepatocytes develop. The liver shrinks and becomes lumpy, which is known as cirrhosis. Cirrhosis progresses slowly and gradually, shutting down the liver's functions. The damage caused by cirrhosis is irreversible, and patients may exhibit symptoms of liver failure. Cirrhosis tends to occur after the age of 50, usually several years after the hepatitis that often accompanies the early stages of the disease. People with cirrhosis caused by NAFLD often also have type 2 diabetes.

[0079] In one embodiment, the method further includes determining the stage of NASH, where the NASH stage is selected from NASH without fibrosis (progressing to fibrosis) or NASH with fibrosis. In some embodiments, NASH induced in association with cirrhosis, such as decompensated cirrhosis or hepatocellular carcinoma (HCC), is also within the scope of the invention.

[0080] The terms "cirrhosis", "liver cirrhosis", or "hepatic cirrhosis" refer to a condition in which the liver does not function properly due to long-term damage. This damage is characterized by replacement of normal liver tissue with scar tissue (i.e., fibrosis). The disease generally develops slowly over months or years and often has no symptoms. Eventually, excessive scarring leads to loss of liver function.

[0081] The term "prognosis prediction" refers to the prediction or likely outcome of a disease. As used herein, this refers to the possible outcomes of a liver disease, including whether the disease (e.g., NASH) will respond to treatment or palliative efforts and / or whether the disease is likely to progress.

[0082] As used herein, the term "progression" can refer to the progression of a disease state. As used herein, the term "regression" can refer to a decrease in the severity of a disease state. When a disease is monitored, this can result in the detection of progression or regression.

[0083] Regression may be due to a change in a health care modality or a therapeutic intervention using a procedure described herein, including, for example, a procedure in a clinical drug trial.

[0084] In one embodiment, the disease stage is early NASH. Unlike NAFL, early NASH is characterized by steatosis, inflammation, high hepatic fat, and hepatocyte injury.

[0085] The method is based on the administration of an exogenous substrate to a subject. An "exogenous substrate" is any compound that can be administered to a subject and metabolized by an enzyme within the subject. The exogenous substrate is a compound recognized by the enzyme of interest, and the enzyme catalyzes the conversion of the substrate into a different compound herein referred to as a "metabolite". The substrate used in the method of the present invention is an exogenous substance, i.e., a xenobiotic. The term xenobiotic refers to a substance that is foreign to the subject's body and is specifically and selectively metabolized by an enzyme. Preferably, the exogenous substance that is converted into a metabolite by an enzyme is also a xenobiotic that is not normally present in the subject's body. In one embodiment, the exogenous substrate is a generally recognized as safe (GRAS) compound.

[0086] The exogenous substrate is selectively metabolized by an enzyme within the subject. This enzyme is one whose activity or expression is downregulated in NASH compared to a healthy subject, or whose activity or expression is upregulated in NASH, i.e., in a patient presenting with the NASH disease. For example, the activity or expression is upregulated or downregulated in the liver tissue of NASH patients compared to healthy subjects. Thus, changes in the expression and / or activity of the enzyme are indicators of NASH.

[0087] For example, a gene encoding an enzyme may be differentially expressed in NASH tissue compared to non-NASH tissue. For example, the enzyme may be expressed at a higher level in NASH tissue compared to non-NASH tissue, or at a lower level in NASH tissue compared to non-NASH tissue. In another embodiment, the enzyme may have a difference in activity in NASH tissue compared to non-NASH tissue. For example, the enzyme may be modified such that its activity is higher or lower in NASH tissue compared to its activity in non-NASH tissue. Gene expression can be measured by techniques known in the art (e.g., by mRNA quantification or measurement of cDNA). The activity of the enzyme can be measured by evaluating its metabolic activity, i.e., the ability of the enzyme to metabolize a substrate.

[0088] Non-NASH tissue can refer to, for example, healthy tissue or NAFLD tissue that has not progressed to NASH, such as tissue from a subject having NAFL, also referred to as steatosis. The tissue may be derived from a particular organ, such as the liver, lung, colon, breast, prostate, etc. In one embodiment, the tissue is liver tissue.

[0089] The method of the present invention may include an additional step of identifying a suitable enzyme whose activity or expression is downregulated in NASH or whose activity or expression is upregulated in NASH compared to non-NASH tissue. The method of the present invention may include a further step of identifying the substrate of the enzyme and, optionally, the metabolite produced by the enzymatic action.

[0090] Accordingly, the method described herein indirectly measures the activity of an enzyme directly related to the NASH disease state in a non-invasive or minimally invasive manner by measuring the activity of the enzyme through the metabolism of a substrate in a biological sample. Based on the enzyme, its ability to break down the substrate, and the association with the NASH disease state, a diagnosis or prognosis can be made regarding the patient's disease state. Based thereon, a suitable treatment can be selected. In particular, the treatment can be selected from one or more of gastric bypass surgery and / or drug-based treatments including the administration of at least one drug selected from statins, incretin mimetics, metformin, rimonabant, thiazolidinediones, and orlistat.

[0091] In one embodiment, the method of the invention includes the step of administering a treatment suitable for treating the NASH disease after the subject has been diagnosed as having NASH or being at risk of developing NASH. Accordingly, the invention also provides a method for treating NASH in a subject, the method including the step of measuring the concentration of an exogenous substrate of an enzyme in a biological sample obtained from the subject and / or the step of measuring the concentration of a metabolite of the substrate, wherein the substrate is a GRAS compound, the enzyme is an aldo-ketoreductase (AKR) or an alcohol dehydrogenase, and the method includes the step of treating the subject.

[0092] The metabolism and conversion of a substrate by one or more enzymes leads to the production of metabolites, i.e., breakdown products that are metabolites. Immediately after supplying the substrate to the subject, the substrate is excreted at high levels in biological matrices such as breath, urine, blood, etc., and as a result of the in vivo conversion of the substrate by the action of one or more enzymes, elimination of the substrate from the biological matrix occurs (washout of the reactant). For example, the kinetic profile of substrate elimination from breath can be used as a readout of the enzyme activity involved in the in vivo conversion of the substrate.

[0093] In addition, the metabolism of a specific substrate through one or more enzymes leads to the production of enzyme-specific metabolites. In this case, the metabolites start at low levels and are excreted into the biological matrix over time as they increase over time due to the in vivo conversion of the substrate by the enzyme. The measurement of such metabolites can be applied as a probe for evaluating the metabolic phenotype of the enzyme involved in the production of the product.

[0094] As further described herein and in the examples, the washout curves for specific metabolites differ between NASH and non-NASH tissues depending on whether the enzyme metabolizing the substrate is overexpressed or downregulated in NASH tissue.

[0095] For example, healthy hepatocytes show a higher production of 2-butanone (by the breakdown of butanol) at the 6-hour time point, and the peak lower area of healthy hepatocytes is larger than that of NASH, which is as expected considering that the alcohol dehydrogenase pathway involved in the conversion of 2-butanol to 2-butanone is downregulated in NASH (see the examples).

[0096] Regarding the production of nonanol by the breakdown of nonanal, NASH hepatocytes showed a higher production of nonanol compared to healthy hepatocytes (the peak area is larger for NASH than for healthy ones). This result is consistent with the overexpression of AKR1B10, the enzyme that converts nonanal to nonanol, observed in NASH livers (see the examples).

[0097] In one embodiment, the enzyme used in the method of the present invention is aldo-ketoreductase (AKR). AKR is an enzyme that catalyzes redox conversions involved in biosynthesis, intermediary metabolism, and detoxification.

[0098] AKR catalyzes redox reactions against a wide variety of substrates, including, among others, glucocorticoids, carbonyl metabolites, glutathione conjugates, and phospholipid aldehydes (Barski, Tipparaju, and Bhatnagar 2008. Aldo-Keto Reductase Superfamily and Its Role in Drug Metabolism and Detoxification. Drug Metabolism Reviews 40 (4)). Considering the wide diversity of biological substrates, AKR seems to have a common function of detoxifying aldehydes and ketones produced by endogenous metabolic reactions, as well as environmental toxins encountered through food, drugs, or other sources (Bachur 1976, Cytoplasmic Aldo-Keto Reductases: A Class of Drug Metabolizing Enzymes. Science 193 (4253): 595-597). Most AKRs use pyridine nucleotides as cofactors to catalyze the reduction of aldehydes and ketones, but they are relatively inefficient alcohol dehydrogenases (Barski, Tipparaju, and Bhatnagar 2008).

[0099] Most of the energy required for carbonyl reduction by AKR is obtained from nucleotide cofactor binding rather than substrate binding, and even when loosely bound to the active site, it results in very efficient reduction of the substrate. This explains the wide range of substrates on which some AKR families, such as the AKR1B family, can act (Grimshaw 1992, Aldose Reductase: Model for a New Paradigm of Enzymic Perfection in Detoxification Catalysts. Biochemistry 31 (42): 10139-10145), and provides the basis for the crucial role of AKR as a detoxifying enzyme. The carbonyl group present in aldehydes is highly reactive and can readily attack nucleophilic centers such as protein amino acids and membrane phospholipids.

[0100] The reduction of aldehyde carbonyls to alcohols by AKR decreases the overall chemical reactivity of the molecule and is one of the mechanisms for the detoxification of reactive aldehydes derived from cells (Barski, Tipparaju, and Bhatnagar 2008). Lipid peroxidation can generate a wide range of toxic aldehydes, because ROS can oxidize any bis-allyl group in the lipid chain (Ayala, Munoz, and ArgOelles 2014, 2014. Lipid Peroxidation: Production, Metabolism, and Signaling Mechanisms of Malondialdehyde and 4-Hydroxy-2-Nonenal. Oxidative Medicine and Cellular Longevity.; Yin, Xu, and Porter 2011.Free Radical Lipid Peroxidation: Mechanisms and Analysis. Chemical Reviews 111(10): 5944 - 5972). Furthermore, aldehydes are also thought to be the main by-products of lipid peroxidation (Barski, Tipparaju, and Bhatnagar 2008). Having both a wide range of substrate specificities and the ability to readily reduce reactive aldehydes, the AKR family is an ideal antioxidant mechanism against lipid peroxidation and ferroptosis.To support this hypothesis, AKR has been shown to reduce products of lipid oxidation such as 4-hydroxynonenal (Gimenez-Dejoz et al., 2015 Substrate Specificity, Inhibitor Selectivity and Structure- Function Relationships of Aldo-Keto Reductase 1: A Novel Human Retinaldehyde Reductase." PLOS ONE 10 (7)), as well as PAPC and POVPC (Srivastava et al., 2004, Aldose Reductase-Catalyzed Reduction of Aldehyde Phospholipids. Journal of Biological Chemistry 279(51)).

[0101] Members of the AKR1B family have been shown to act on several highly volatile compounds. Indeed, AKR1B1, AKR1B10, and AKR1B15 have shown substrate specificity for, among others, the volatile aldehydes benzaldehyde and cinnamaldehyde, the alkanal hexanal, the alkenal 4-hydroxynonenal, hexenal, and farnesal, the ketone 3-nonen-2-one, and the dicarbonyls 2,3-butanedione and 2,3-hexanedione (Gimenez-Dejoz et al., 2015).

[0102] Aldo-keto reductase family 1 member B10 (AKR1B10) is associated with HCC and is secreted into the bloodstream by hepatocytes via a lysosome-mediated non-classical pathway. Secretion of the AKR1B10 protein is associated with advanced NASH (Kanno, M. et al., 2019. Serum aldo-keto reductase family 1 member B10 predicts advanced liver fibrosis and fatal complications of nonalcoholic steatohepatitis. J Gastroenterol 54, 549-557).

[0103] In one embodiment, the enzyme may be a member of the AKR family 1, and in particular, may be a member of the AKR family 1 member B10 (AKR1B10).

[0104] In another embodiment, the enzyme used in the method of the present invention is alcohol dehydrogenase. Alcohol metabolism is a well-characterized biological process mediated by the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) families.

[0105] Alcohol dehydrogenase catalyzes the oxidation of primary and secondary alcohols to the corresponding aldehydes or ketones. Changes in the alcohol metabolism process in response to the progression of human NASH have been investigated, and the activity and expression of alcohol dehydrogenase enzymes have been examined (Li, H., Toth, E. & Cherrington, N. J. Alcohol Metabolism in the Progression of Human Nonalcoholic Steatohepatitis. Toxicol Sci 164, 428-438, (2018)).

[0106] In one embodiment, the enzyme is αα - alcohol dehydrogenase (ααADH). In one embodiment, the enzymes are ALDH4A1, ADH1A, ADH1B, ADH4, and ALDH2.

[0107] The exogenous substrate used in the method of the present invention is specific to the enzyme such that the substrate is selectively metabolized by the enzyme. Thus, the exogenous substrate can be any substrate suitable for detecting enzyme activity. In one embodiment, the substrate and / or its metabolite is a VOC secreted into the biological matrix, preferably a VOC secreted into the biological matrix at a high rate. Generally, VOCs are defined as organic compounds whose composition allows them to evaporate under normal indoor atmospheric conditions of temperature and pressure. Since the volatility of a compound is generally higher the lower its boiling point temperature, the volatility of an organic compound is defined and may be classified by its boiling point. Volatile compounds are, for example, compounds secreted by the human body into a gaseous fluid including exhaled breath, skin emissions, etc. Optionally, the substrate and / or metabolite is a VOC that can be measured in the biological matrix without using a label such as an isotope label. Preferably, the exogenous substrate can be selected from aldehydes and / or alcohols.

[0108] According to various aspects of the present invention, in one embodiment, the substrate may be a VOC and the concentration of the exhaled VOC substrate in the exhaled breath is measured.

[0109]

[0110] ​In one embodiment, the substrate is a VOC and its metabolite is not a VOC. In another embodiment, the substrate is not a VOC and its metabolite is a VOC. In this case, the concentration of the metabolite in the exhaled breath is measured. In another embodiment, the substrate is a VOC and its metabolite is a VOC. In this case, the concentration of the substrate and / or metabolite in the exhaled breath is measured.

[0111] If the substrate is a VOC, it may or may not be labeled.

[0112] The VOCs measured according to the method need not be naturally present in / produced by the subject and need not be excreted into the biological matrix. This ensures that any readings are not subject to contamination by endogenous VOCs that are naturally produced and found in the biological matrix.

[0113] In one embodiment, the substrate is a compound that is naturally occurring (but not endogenously produced), for example, a food compound. This has the advantage that it can be provided to the subject without causing side effects. In one embodiment, the substrate has no therapeutic benefit. In one embodiment, the substrate is not a compound that does not occur naturally.

[0114] In one embodiment, the substrate is a GRAS compound, for example, a GRAS compound that is a VOC. "GRAS" is the acronym for Generally Recognized As Safe. Under sections 201(s) and 409 of the Federal Food, Drug, and Cosmetic Act, any substance intentionally added to food is a food additive and is subject to premarket review and approval by the FDA unless it is generally recognized among qualified experts as being safe under the conditions of its intended use or its use is otherwise excluded from the definition of a food additive. For example, a GRAS compound can be a naturally occurring compound. For example, a GRAS compound can be selected from food or food additives. In one embodiment, the GRAS compound is a vitamin, a phenolic flavorant, a natural oil, an alcohol, an amino acid, or an antioxidant. In one embodiment, the GRAS compound is a plant extract. In one embodiment, the GRAS compound is a plant-based substance mainly used for flavoring, coloring, or preserving food. In one embodiment, the GRAS compound is an aliphatic or aromatic terpene hydrocarbon or a terpenoid. In one embodiment, the GRAS compound is a food flavor approved in the EU.

[0115] In one embodiment, the substrate is not a VOC and its metabolite is not a VOC. In that embodiment, the substrate is a labeled reactant and the labeled reactant and / or the labeled metabolite can be measured in exhaled breath. The label can be an isotope label, for example, 12C, 13C, 14C, 2H, 14N, or 18O.

[0116] In one embodiment, the substrate and / or the metabolite is a VOC and the substrate is not labeled. Thus, the substrate and / or the metabolite can be measured in a biological matrix without using a label, so a label is not necessary.

[0117] In one embodiment, the enzyme is an AKR enzyme. In one embodiment, the enzyme is AKR1B10. In one embodiment, the substrate is selected from aldehydes and / or alcohols. In one embodiment, the enzyme is an AKR enzyme such as AKR1B10, the substrate is nonanal, and the metabolite is nonanol. When nonanal is used in the method of the present invention, 1-nonanal can be used. Since 1-nonanal can be metabolized to 1-nonanol, 1-nonanol can be the metabolite detected in the method.

[0118] In one embodiment, the enzyme is an AKR enzyme such as AKR1B10, the substrate is trans-2-hexenal, and the metabolite is trans-2-hexenol.

[0119] In one embodiment, the enzyme is an AKR enzyme such as AKR1B10, the substrate is hexanal, and the metabolite is hexanol.

[0120] In one embodiment, the enzyme is an AKR enzyme such as AKR1B10, the substrate is benzaldehyde, and the metabolite is benzyl alcohol.

[0121] In one embodiment, the enzyme is an AKR enzyme such as AKR1B10, the substrate is citral, and the metabolite is nerol.

[0122] In yet another embodiment, the enzyme used in the method of the present invention is cytochrome P450 (CYP). CYP is a superfamily of enzymes that generally function as monooxygenases. CYP contains a heme cofactor and is a catalyst in steroid hormone synthesis and drug metabolism.

[0123] In one embodiment, the enzyme is CYP1A1, CYP1A2, CYP1B1, CYP2, CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP2W1, CYP3, CYP3A4, CYP3A5, CYP3A7 or CYP3A43. In one embodiment, the enzyme is CYP2C19, CYP2C9 and / or CYP3A4.

[0124] In one embodiment, the enzyme is a CYP enzyme, the substrate is 2-butanone, and the metabolite is 3-hydroxy-2-butanone and / or 2,3-butanediol.

[0125] In one embodiment, the enzyme is a CYP enzyme, the substrate is benzoic acid, and the metabolite is hippuric acid. Saltzman A, Caraway WT. Cinnamic acid as a test substance in the evaluation of liver function. J Clin Invest 1953;32(8):711-719 demonstrates the conversion of benzoic acid to hippuric acid in Figure 7 of that publication. In one embodiment, the enzyme is glycine N-acyltransferase. In one embodiment, the substrate is benzoic acid and the metabolite is hippuric acid.

[0126] In one embodiment, the enzyme is a CYP enzyme, the substrate is 2-pentanone, and the metabolite is 2,3-pentanediol.

[0127] In one embodiment, the substrate can produce a first metabolite, and / or the first metabolite can be used as a substrate for producing a second metabolite. In some embodiments, the concentration of the substrate can be measured. In some embodiments, the concentration of the first metabolite can be measured. In some embodiments, the concentration of the second metabolite can be measured. In some embodiments, the concentrations of the substrate and the first metabolite can be measured. In some embodiments, the concentrations of the first metabolite and the second metabolite can be measured. In some embodiments, the concentrations of the substrate and the second metabolite can be measured. In some embodiments, the concentrations of the substrate, the first metabolite, and the second metabolite can be measured.

[0128] In one embodiment of the above method, the concentration of the substrate is measured. In another embodiment of the above method, the concentration of the metabolite is measured. For example, the concentrations of metabolites such as nonanol, trans-2-hexenol, hexanol, benzyl alcohol, nerol, 3-hydroxy-2-butanone, 2,3-butanediol, benzoic acid, hippuric acid, 2-pentanone and 2,3-pentanediol are measured. In one embodiment of the above method, the concentrations of the substrate and the metabolite are measured.

[0129] In the above method, the concentration of one or more substrates and / or one or more metabolites is measured. Thus, in one embodiment, the concentrations of multiple metabolites can be measured.

[0130] Thus, the method provided herein enables the examination of multiple compounds in exhaled breath. This enables the examination for the presence of two or more types of diseases. Further, multiple compounds specific to a particular type of disease can be measured in exhaled breath, thereby enabling a more accurate diagnosis based on multiple parameters to be evaluated. Thus, in one embodiment, the present invention relates to a method for detecting a disease, the method comprising the steps of measuring the concentration of two or more exogenous substrates for the enzyme and / or evaluating the activity of one or more disease-specific enzymes by measuring the concentration of two or more metabolites of the substrate in the exhaled breath of the subject.

[0131] In one embodiment, the method for detecting liver diseases disclosed herein includes the steps of measuring the concentrations of two or more exogenous substrates of two or more enzymes in the breath of a subject and / or measuring the concentrations of two or more metabolites of the substrates to evaluate the activities of the two or more enzymes. Accordingly, the method described herein can be a multiplex method that enables simultaneous evaluation of multiple enzyme activities in the same breath sample.

[0132] Therefore, in one aspect, the present invention is a method for detecting or predicting the prognosis of NAFLD that has progressed to the stage of NASH in a subject, including the steps of measuring the concentration of an exogenous substrate of an NASH-specific enzyme and / or measuring the concentration of a metabolite of the substrate in a biological sample obtained from the subject, wherein the substrate is nonanal. Preferably, the present invention is a method for detecting or predicting the prognosis of NAFLD that has progressed to the stage of NASH in a subject, including the steps of measuring the concentration of an exogenous substrate of AKR1B10 in a biological sample obtained from the subject and / or measuring the concentration of a metabolite of the substrate, wherein the substrate is nonanal. Further, since nonanal can be metabolized to nonanol, in one embodiment, the metabolite is nonanol.

[0133] In one embodiment, the enzyme is an alcohol dehydrogenase such as ααADH. In one embodiment, the substrate is selected from aldehydes and / or alcohols. In one embodiment, the enzyme is alcohol dehydrogenase, the substrate is butanol, and the metabolite is butanone.

[0134] In one embodiment, the enzyme is alcohol dehydrogenase, the substrate is 2-pentanone, and the metabolites are 2-pentanol, 3-hydroxy-2-pentanone, and / or 2,3-pentanediol.

[0135] In one embodiment, the enzyme is alcohol dehydrogenase, the substrate is benzyl alcohol, and the metabolites are benzaldehyde and / or benzoic acid.

[0136] In one embodiment, the enzyme is aldehyde dehydrogenase, the substrate is benzaldehyde, and the metabolite is benzoic acid.

[0137] The present invention also relates to a method for detecting or predicting the prognosis of NASH in a subject, comprising the step of measuring the concentration of an exogenous substrate of an enzyme in a biological sample obtained from the subject, and / or the step of measuring the concentration of a metabolite of the substrate, wherein the substrate is a GRAS compound, the substrate is 2-pentanone, and the metabolites are 2-pentanol, 3-hydroxy-2-pentanone and / or 2,3-pentanediol.

[0138] Therefore, in one aspect, the present invention relates to a method for detecting or predicting the prognosis of NAFLD that has progressed to the stage of NASH in a subject, comprising the step of measuring the concentration of an exogenous substrate of a NASH-specific enzyme, and / or the step of measuring the concentration of a metabolite of the substrate in a biological sample obtained from the subject, wherein the substrate is butanol. Preferably, the present invention relates to a method for detecting or predicting the prognosis of NAFLD that has progressed to the stage of NASH in a subject, comprising the step of measuring the concentration of an exogenous substrate of ADH in a biological sample obtained from the subject, and / or the step of measuring the concentration of a metabolite of the substrate, wherein the substrate is butanol. Further, since butanol can be metabolized to butanone, in one embodiment, the metabolite is butanone.

[0139] When butanol is used in the method, 2-butanol may be used. Since 2-butanol can be metabolized to 2-butanone, 2-butanone can be a metabolite detected in the method.

[0140] In the above method, the concentration of one or more substrates and / or one or more metabolites is measured. Thus, in one embodiment, the concentration of a plurality of metabolites can be measured.

[0141] By using a hepatocyte model as described in the examples which is a model of early liver disease, the inventors have developed a method that enables distinguishing NASH from other stages of liver disease and thus enables early diagnosis and intervention. This distinction relies on changes in enzyme activity induced by NASH that result in changes in the metabolic rate of specific compounds. Quantification of these compounds enables the identification of subjects having NASH.

[0142] In another aspect, the present invention relates to a method for distinguishing NASH from other stages of NAFLD in a subject, comprising the step of measuring the concentration of an exogenous substrate of an enzyme in a biological sample obtained from the subject and / or the step of measuring the concentration of a metabolite of the substrate, wherein the substrate is a GRAS compound.

[0143] In another aspect, the present invention relates to a method for detecting or predicting the prognosis of early NASH in a subject, comprising the step of measuring the concentration of an exogenous substrate of an enzyme in a biological sample obtained from the subject and / or the step of measuring the concentration of a metabolite of the substrate, wherein the substrate is a GRAS compound.

[0144] In another aspect, the present invention relates to a method for determining the stage of NASH in a subject, comprising the step of measuring the concentration of an exogenous substrate of an enzyme in a biological sample obtained from the subject and / or the step of measuring the concentration of a metabolite of the substrate, wherein the substrate is a GRAS compound.

[0145] As described above, in these methods, the activity or expression of said enzyme is upregulated or downregulated in NASH. For example, the enzyme may be AKR or ADH as further described above. For example, the enzyme may be AKR, and the substrate and metabolite may be selected from those described above. For example, the substrate may be selected from nonanal, trans-2-hexenal, hexanal, benzaldehyde, citral, and the corresponding metabolites from each of nonanol, trans-2-hexenol, hexanol, benzyl alcohol, nerol. For example, the enzyme may be ADH, and the substrate and metabolite may be selected from those listed above. For example, the substrate may be selected from butanol or 2-pentanone, and the corresponding metabolites may be selected from butanone, 2-pentanol, 3-hydroxy-2-pentanone or 2,3-pentanediol, respectively.

[0146] In another embodiment, the enzyme is a CYP enzyme. The cytochrome CYP450 (CYP450) enzyme family is involved in the metabolism of most drugs and lipophilic xenobiotics and is thus of great importance for clinical pharmacology. Several different families of CYP450 enzymes are present in the human body, but the enzymes belonging to the 1-, 2-, and 3-families are involved in the metabolism of most administered therapeutic drugs.

[0147] These enzymes convert prodrugs into the corresponding bioactive compounds and active drugs into inactive metabolites, which are then excreted from the human body. Differences in the enzyme activity of these enzymes lead to differences in the in vivo conversion of xenobiotics and ultimately result in the toxicity of the compound or the ineffectiveness of the drug. Many factors contribute to the diversity of CYP450 activity and their resulting metabolic efficiency.

[0148] In one embodiment, the CYP450 enzyme is selected from family 1, 2, or 3. For example, the CYP450 enzyme is selected from CYP1A1, CYP1A2, CYP1B1, CYP2, CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP2W1, CYP3, CYP3A4, CYP3A5, CYP3A7, or CYP3A43. In one embodiment, the enzyme is CYP2C19, CYP2C9, and / or CYP3A4. In another embodiment, the liver enzyme is selected from glutathione S-transferase, arylsulfatase, and UDP-glucuronyltransferase or aldehyde dehydrogenase.

[0149] In one embodiment, the enzyme is CYP2C19 and / or CYP2C9, and the substrate is limonene. In one embodiment, the liver enzyme is CYP2C19 and / or CYP2C9, the substrate is limonene, and the metabolite is perillyl alcohol. In one embodiment, the liver enzyme is CYP3A4, and the substrate is eucalyptol.

[0150] In one embodiment, the enzyme is glycine N-acyltransferase. In one embodiment, the enzyme is glycine N-acyltransferase, the substrate is benzoic acid, and the metabolite is hippuric acid.

[0151] This method requires a step of measuring the concentration of an exogenous substrate and / or its metabolite in a biological sample from a subject, i.e., the sample to be tested. The term biological sample can be used interchangeably with the term biological matrix. In one embodiment, the biological sample or matrix is selected from exhaled breath, urine, blood, serum, and / or tissue. The biological sample can be a tissue sample such as adipose tissue, liver, brain, bone marrow, muscle, or hair. In one embodiment, the biological sample is a sample of body fluid. Methods for obtaining a body fluid sample are well known in the art. In one embodiment, the body fluid sample can be a sample of blood, urine, or exhaled breath. The blood sample can include one or more of plasma, red blood cells, white blood cells, and platelets. The blood sample can include any combination of plasma, red blood cells, white blood cells, and platelets.

[0152] In one embodiment, the sample is exhaled breath.

[0153] When the body fluid sample is a sample of exhaled breath, the exhaled breath sample can include air exhaled from one or more different parts of the subject's body (e.g., nostrils, pharynx, trachea, bronchioles, alveoli, etc.). For the method of collecting and measuring the exhaled breath sample, the devices and methods described in WO2017 / 187120 or WO2017 / 187141 (both publications are incorporated herein by reference) can be used.

[0154] In an embodiment where the biological sample is a sample of exhaled breath, this can be obtained by collecting exhaled breath from the subject, for example, by asking the subject to exhale air into a gas sampling container such as a bag, bottle, or any other suitable gas sampling product. Preferably, the gas sampling container resists gas permeation to and from the outside of the bag and / or is chemically inert, thereby ensuring the integrity of the sample. Also, exhaled breath can be collected using an exhaled breath collection device. Preferably, the collection of the exhaled breath sample is performed by a minimally invasive or non-invasive method.

[0155] Determination of the amount of one or more VOCs in a breath sample from a subject can be done by use of at least one technique including, but not limited to, gas chromatography (GC), gas chromatography-mass spectrometry (GC / MS), liquid chromatography-tandem mass spectrometry (LC / MS), ion mobility spectrometry / mass spectrometry (IMS / MS), proton transfer reaction mass spectrometry (PTR-MS), electronic nose devices, quartz crystal microbalance or chemosensitive sensors.

[0156] The amount of one or more VOCs in a breath sample from a subject can be determined using thermal desorption-gas chromatography-time of flight mass spectrometry (GC-TOF-MS). In certain embodiments, the subject's breath is collected in an inert bag, and subsequently the contents of the bag are transported to a desorption tube under standardized conditions, and the VOCs are analyzed by thermally desorbing the contents of the tube, followed by separation by capillary gas chromatography. Subsequently, the volatile organic peaks are detected by MS and identified using a library such as the National Institute of Standards and Technology. Thermal desorption can be performed at the inlet of the GC, for example, at a temperature of about 200-350 °C. In all chromatography, separation occurs when the sample mixture is introduced (injected) into the mobile phase. Gas chromatography (GC) typically uses an inert gas such as helium as the mobile phase. GC / MS enables the separation, identification, and / or quantification of individual components from a biological sample. Examples of MS methods that can be used with the present invention include, but are not limited to, electron ionization, electrospray ionization, glow discharge, field desorption (FD), fast atom bombardment (FAB), thermospray, desorption / ionization on silicon (DIOS), direct analysis in real time (DART), atmospheric pressure chemical ionization (APCI), secondary ion mass spectrometry (SIMS), spark ionization, and thermal ionization (TIMS). Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) is an example of a mass spectrometry method that can be used to determine one or more VOCs from a breath sample from a subject.

[0157] In one embodiment, the method includes collecting a plurality of different selected breath samples or fractions thereof on a single breath sample capture device, and the method (a) collecting a first breath sample by contacting the sample with a capture device comprising an adsorbent material, (b) A step of collecting a second breath sample by bringing a second sample into contact with the capture device, wherein the first and second breath samples are captured on the capture device in a spatially separated manner comprises.

[0158] Accordingly, the present invention also (a) A step of administering the substrate described herein, (b) A step of collecting a first breath sample by bringing a sample into contact with a capture device containing an adsorbent material, and (c) A step of collecting a second breath sample by bringing a second sample into contact with the capture device, wherein the first and second breath samples are captured on the capture device in a spatially separated manner relates to a method comprising.

[0159] In some embodiments, the capture device comprises an adsorbent material in the form of a porous polymer resin. Suitable adsorbent materials include Tenax® resin and Carbograph® material. Tenax® is a porous polymer resin based on 2,6-diphenyl-p-propylene oxide monomer. Carbograph® material is graphitized carbon black. In one embodiment, the material is Tenax GR, which comprises a mixture of Tenax® TA and 30% graphite. One of the Carbograph® adsorbents is Carbograph 5TD. In one embodiment, the capture device comprises both Tenax GR and Carbograph 5TD. Advantageously, the capture device is an adsorption tube. These are hollow metal cylinders, typically of standard dimensions (length 3 + 1 / 2 inches, inner diameter 1 / 4 inch), filled with a suitable adsorbent material.

[0160] A non-NASH subject is a subject who does not have NASH, but may or may not have NAFL. Non-NASH subjects include healthy subjects. As used herein, "healthy subject" refers to a subject who does not have the NASH disease of interest, i.e., as defined for humans.

[0161] As used herein, "reference value" means a value determined by performing an assay method on a plurality of subjects of interest. The subjects of interest can be healthy subjects or subjects diagnosed with a certain disease.

[0162] "Probability of disease state" means that the probability that the disease state is present in the subject sample is about 50% or more, for example, 60%, 70%, 80% or 90%.

[0163] As used herein, a decrease or increase can be 5%, 10%, 20%, 30%, 40%, 50% or more, or at least or about 2-fold, at least or about 2.5-fold, at least or about 3-fold, at least or about 3.5-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, or at least or about 10-fold.

[0164] The method of the present invention includes the steps of determining the concentration of a substrate and / or metabolite in an exhaled sample of a subject to be tested, and subsequently comparing that concentration to a reference / baseline value or range. Typically, the reference / baseline value represents the concentration of the substrate and / or metabolite in a healthy person or non-NASH subject who does not have NASH or is not expected to develop NASH.

[0165] The sample can be obtained at any time. The sample can be obtained at any time before or after administration of the substrate, for example, about 10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours, about 15 hours, about 18 hours, about 20 hours, about 22 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 3 years, about 5 years or more before or after the start of a treatment or therapy. This time point can be earlier or later.

[0166] Variations (either upward or downward) in the levels of substrates and / or metabolites from a baseline / reference value or range indicate that the patient has NASH, an increased risk of NASH, and / or an increased risk of long-term mortality. For example, in embodiments where the enzyme is AKR, an increase in the concentration of metabolites in the breath compared to the baseline value in healthy individuals indicates a risk of NASH. The AKR enzyme is overexpressed in NASH disease, and therefore the conversion of the substrate to the metabolite is increased. Conversely, a decrease in the level of the substrate in the breath indicates a risk of NASH.

[0167] For example, in embodiments where the enzyme is ADH, the alcohol dehydrogenase pathway involved in the conversion of 2-butanol to 2-butanone is downregulated in NASH, and therefore the production of 2-butanone is higher in healthy subjects than in NASH patients.

[0168] The algorithms used to calculate the risk assessment score in the methods disclosed herein may group concentration values of substrates and / or metabolites, and the risk score can be derived from any algorithm known in the art. An algorithm is a set of rules for describing the risk assessment of NASH. The set of rules may be defined algebraically exclusively, but may also include alternative or multiple decision points that require domain-specific knowledge, expert interpretation, or other clinical indicators. Many algorithms can be developed that can provide different risk assessments using suitable concentration profiles of substrates and / or metabolites. For example, an individual's risk score may be generated using a Cox proportional hazards model. The prognostic category classification of an individual can also be determined by using a statistical model or machine learning algorithm that calculates the probability of recurrence based on the individual's concentration of substrates and / or metabolites.

[0169] Based on the determination of risk, an individual can be assigned to a risk group (e.g., a tertile group or a quartile group) based on a selected value of the risk score, and all individuals having values within a given range can be classified as belonging to a particular risk group. Thus, the selected values define risk groups of patients having higher or lower risks, respectively. The risk groups can be further classified according to different ranges of mortality, e.g., 6-month, 1-year, 2-year, 3-year, 4-year, 5-year, 10-year, 25-year mortality. The risk groups can be further classified according to different ranges of events associated with NASH, which may include but are not limited to the likelihood of progression to NASH with fibrosis or NASH with cirrhosis.

[0170] The concentration of the substrate and / or metabolite can be measured using methods known in the art. Concentration, as used herein, means the content or mass of the substrate and / or metabolite in a biological sample, such as expressed in grams per liter (g / l). In one embodiment, the concentration is measured over time, for example, by measuring the kinetics of elimination. For example, the concentration is measured by evaluating the kinetic profile of elimination of the substrate from exhaled breath and subsequently using it as a readout. Additionally or alternatively, the secretion of metabolites that may be derived from the substrate can be measured over time. For example, both the elimination of the substrate and the secretion of metabolites from a biological sample can be measured simultaneously or at different time points in the same biological sample.

[0171] In one embodiment, the concentration or amount of the substrate and / or its metabolite can be determined absolutely or relatively in a plurality of biological samples, e.g., a first exhaled breath sample (collected during a first period) and a second and / or further exhaled breath sample (collected during a subsequent second or further period), and thus analysis of the kinetics or rate of change of its concentration over time is possible.

[0172] In some embodiments, when the biological sample is an exhaled breath sample, the capture device includes an adsorbent material in the form of a porous polymer resin. Suitable adsorbent materials include Tenax® resin and Carbograph® material. Tenax® is a porous polymer resin based on 2,6-diphenyl-p-propylene oxide monomer. Carbograph® material is graphitized carbon black. In one embodiment, the material is Tenax GR, which includes a mixture of Tenax® TA and 30% graphite. One of the Carbograph® adsorbents is Carbograph 5TD. In one embodiment, the capture device includes both Tenax GR and Carbograph 5TD. Advantageously, the capture device is an adsorption tube. These are hollow metal cylinders, typically of standard dimensions (length 3 + 1 / 2 inches, inner diameter 1 / 4 inch) filled with a suitable adsorbent material.

[0173] In one embodiment, the method of the present invention further includes establishing a value to be tested for the concentration of one or more substrates and / or metabolites. The value to be tested can be compared with one or more reference (control) values, and the possibility of NASH is indicated by the difference between the value to be tested and the reference value.

[0174] In one embodiment, the reference value is from a non-NASH subject, such as a healthy subject.

[0175] In another embodiment, the reference value is from a subject diagnosed with NASH (with or without fibrosis), or a subject in whom NASH has progressed to cirrhosis or HCC.

[0176] The reference level of the test compound (i.e., the substrate or metabolite) can be determined by determining the level of the test compound in a sufficiently large number of samples obtained from normal healthy control subjects to obtain a predetermined reference value or threshold value. The reference level can also be determined by determining the level of the test compound in a sample from the patient before treatment.

[0177] In one embodiment, the method of the present invention further includes a step of comparing the value of the subject with one or more reference values. In one embodiment, the reference value is from a non-NASH subject, for example, a healthy subject. In another embodiment, the reference value is from a subject diagnosed with NASH. In another embodiment, the reference value is the value of a subject diagnosed with non-alcoholic fatty liver (NAFL). In another embodiment, the reference value is the value of a subject who has progressed to cirrhosis or HCC.

[0178] In one embodiment, the reference value is the value of a NAFL subject corresponding to a value calculated from a NAFL subject. In one embodiment, the presence of one or more subject values that are greater than each range of healthy subject values indicates a substantial likelihood of NASH disease state in the subject under examination.

[0179] In one embodiment, the reference value is the value of a healthy subject corresponding to a value calculated from a healthy subject. In one embodiment, the presence of one or more subject values that are greater than each range of healthy subject values indicates a substantial likelihood of NASH disease state in the subject under examination.

[0180] In one embodiment, compared with the reference value, for example, an increase in the concentration of a test compound, such as a substrate or metabolite, by 5%, 10%, 20%, 30%, 40%, 50% or more indicates a diagnosis of NASH or the risk that the subject will develop NASH.

[0181] In one embodiment, compared with the reference value, for example, a decrease in the concentration of a test compound, such as a substrate or metabolite, by 5%, 10%, 20%, 30%, 40%, 50% or more indicates a diagnosis of NASH or the risk that the subject will develop NASH.

[0182] In one embodiment, when an appropriate reference serves as an indicator for a subject without NASH, a detectable difference (e.g., a statistically significant difference) between a value determined from a subject in need of NASH characterization or diagnosis and the appropriate reference may serve as an indicator of NASH in the subject. In one embodiment, when an appropriate reference serves as an indicator of NASH, the absence of a detectable difference (e.g., the absence of a statistically significant difference) between a value determined from a subject in need of NASH characterization or diagnosis and the appropriate reference may serve as an indicator of NASH in the subject. In one embodiment, an increase in the concentration of a metabolite compared to a reference sample serves as an indicator of NASH.

[0183] Thus, in one aspect, the method includes detecting the concentration of a substrate and / or metabolite in the breath from a subject, and diagnosing the subject as having an increased likelihood or risk of a NASH disease state when the level of one or more of the substrate and / or metabolite is different from that of a healthy reference subject.

[0184] Thus, any of the methods described herein may further include the following steps: a) comparing the amount of one or more VOCs in a biological sample to a reference value, wherein the reference value represents a known diagnosis, prognosis prediction, and / or monitoring status of NASH, b) finding a deviation or the absence of a deviation of the amount of the one or more VOCs from the reference value, and c) assigning the finding of deviation or no deviation to a specific diagnosis, prognosis prediction, and / or monitoring status of NASH in the subject.

[0185] The term "quantitative deviation" refers to either an increase or a decrease in the amount of one or more VOCs in a biological sample from a subject, compared to a reference value. An "increased amount" means that the amount of said one or more VOCs in the biological sample from the subject is statistically higher than the reference value. A "decreased amount" means that the amount of said one or more VOCs in the biological sample from the subject is statistically lower than the reference value. An amount can be considered statistically higher or lower if its value is different from a predetermined threshold value. This threshold value can be, for example, the median of the amount of VOCs determined in biological samples from a population of healthy subjects.

[0186] The term "no quantitative deviation" refers to the amount of one or more VOCs of the present invention in a breath sample from a subject being of the same degree or unchanged, compared to a reference value. A "same degree or unchanged level" means that the difference in the amount of said one or more VOCs in the biological sample from the subject, compared to the reference value, is not statistically significant. Preferably, the reference value is obtained in a breath sample from one or more subjects of the same species, the same gender and the same age group as the subject for whom NASH is being determined, prognosed or monitored. Alternatively, the reference value may be a previous value for the amount of one or more VOCs obtained in a breath sample from a particular subject. This type of reference value can be used when the method is used, for example, to monitor NASH over time or to monitor the response of a subject to a particular treatment.

[0187] The method may also include determining a risk score for the subject based on the concentration of metabolites and / or substrates in the sample, and providing a prognosis prediction for the subject using the risk score, wherein the risk score serves as an indicator for the prognosis prediction.

[0188] The method may include determining the concentration of two or more exogenous substrates of NASH-specific enzymes and / or determining the concentration of two or more metabolites of said substrates.

[0189] In the method of the present invention, the subject is administered an exogenous substrate of a NASH-specific enzyme. In certain embodiments, the method can include the step of administering an exogenous substrate of a NASH-specific enzyme to the subject. Administration of the substrate can be via any suitable route including, but not limited to, oral, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitreal, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. Preferably, the substrate is administered orally. Once the substrate is administered to the subject, the concentration of the substrate and / or its metabolite in a biological sample obtained from the subject can be determined.

[0190] One aspect is a method of monitoring the progression of NASH in a subject, comprising the step of measuring the concentration of an exogenous substrate of a NASH-specific enzyme and / or the concentration of a metabolite of said substrate in a biological sample obtained from the subject, wherein said substrate and / or its metabolite is a VOC, the substrate is a GRAS compound, and the enzyme is the above-mentioned aldo-keto reductase (AKR) or alcohol dehydrogenase.

[0191] One aspect is a method for determining the effectiveness of treatment, comprising the step of evaluating the activity of an enzyme in a subject diagnosed with NASH by measuring the concentration of an exogenous substrate of said enzyme and / or the concentration of a metabolite of said substrate in a biological sample obtained from the subject, wherein said subject has received treatment for NASH and the enzyme is the above-mentioned aldo-keto reductase (AKR) or alcohol dehydrogenase.

[0192] A method for determining the effectiveness of a treatment may include the following steps: (a) obtaining a first sample from a patient before the start of the treatment or therapy (or at a first time point after the start of the treatment or therapy, or when the treatment or therapy is initiated); (b) assaying the levels of substrates and / or metabolites in the first sample; (c) obtaining a second sample from the patient after the start of the treatment or therapy (or at a second time point after the start of the treatment or therapy); (d) assaying the levels of substrates and / or metabolites in the second sample; and (e) comparing the levels of substrates and / or metabolites in the first sample with the levels of substrates and / or metabolites in the second sample.

[0193] Depending on the substrate and / or metabolite being assayed, if its level increases or decreases compared to the level of the substrate and / or metabolite obtained in the first sample, the therapy is considered effective. An effective treatment or therapy may be continued or discontinued if the patient's condition improves and treatment is no longer required. An ineffective treatment may be changed or modified, or replaced with another treatment.

[0194] The treatment may include surgery or at least one drug selected from statins, incretin mimetics, metformin, rimonabant, thiazolidinediones, and orlistat.

[0195] To determine the effectiveness of a treatment, multiple samples can be obtained at various time points. Thus, the method may include analyzing a first biological sample obtained from the subject at a first time point and subsequently analyzing one or more additional biological samples obtained from the subject at one or more additional time points, or their ratios.

[0196] Thus, the method may also include administering the treatment.

[0197] In one embodiment, the treatment of NASH is a drug-based treatment including gastric bypass surgery and / or administration of at least one drug selected from statins, incretin mimetics, metformin, rimonabant, thiazolidinediones, and orlistat. The treatment of NASH is known in the art (Ganguli et al., Hepat Med. 2019; 11: 159-178).

[0198] In one aspect of any of the methods described, the substrate is not limonene. In one aspect of any of the methods described, the substrate is not a CYP enzyme.

[0199] In some embodiments, the techniques described herein are associated with a programmable machine designed to perform a series of arithmetic or logical operations as provided by the methods described herein. For example, some embodiments of the present technology are associated with (e.g., implemented in) computer software and / or computer hardware. In one aspect, the present technology relates to a computer comprising a form of memory, elements for performing arithmetic and logical operations, and a processing element (e.g., a microprocessor) for executing a series of instructions (e.g., the methods provided herein) for reading, manipulating, and storing data. Accordingly, certain embodiments employ a process that includes data stored in or transferred through one or more computer systems or other processing systems. Embodiments also relate to an apparatus for performing these operations. This apparatus can be specially constructed for the required purpose or can be a general-purpose computer (or group of computers) selectively activated or reconfigured by a computer program and / or data structure stored in the computer. In some embodiments, a group of processors jointly and / or in parallel perform some or all of the recited analysis operations (e.g., via network computing or cloud computing).

[0200] In some embodiments, the microprocessor is part of a system for determining the presence of one or more mRNAs or miRNAs associated with liver disease, creating a standard curve, determining the specificity and / or sensitivity of an assay or marker, calculating an ROC curve, and performing sequence analysis, all of which are described herein or known in the art.

[0201] In some embodiments, the microprocessor is part of a system for determining the amount, e.g., concentration, of one or more substrates and / or metabolites associated with NASH, creating a standard curve, determining the specificity and / or sensitivity of an assay or marker, calculating an ROC curve, and performing sequence analysis, all of which are described herein or known in the art. The amount of one or more substrates and / or metabolites can be determined by the abundance measured per mole or millimole.

[0202] The amount of one or more substrates and / or metabolites can be determined by assays known to those skilled in the art and described herein, including measurements using optical signals or other measurements known to those skilled in the art.

[0203] In some embodiments, the microprocessor or computer uses an algorithm to measure the amount of one or more substrates and / or metabolites. The algorithm can include mathematical interactions between marker measurements or mathematical transformations of marker measurements. The mathematical interactions and / or mathematical transformations can be presented in a linear, non-linear, discontinuous, or discrete manner.

[0204] In some embodiments, software or hardware components receive the results of multiple assays and determine a single-value result for reporting to a user that indicates the NASH disease risk based on the results of the multiple assays. Related embodiments calculate risk factors based on mathematical combinations (e.g., weighted combinations, linear combinations) of results from multiple assays as described elsewhere herein.

[0205] Compositions and Kits The present invention relates to a kit for detecting, diagnosing, screening or predicting the prognosis of NASH, for distinguishing NASH from NAFL, or for determining the effectiveness of NASH treatment, comprising nonanal and / or butanol and a device for capturing a biological sample from a patient.

[0206] The kits described herein may include a substrate, i.e., a composition for administration comprising nonanal, butanol, trans-2-hexenal, hexanal, benzaldehyde, citral, or 2-pentanone or limonene. This can be formulated as an oral dosage form, for example, as a tablet or capsule.

[0207] The kit may include instructions for evaluating or monitoring NASH in a patient based on the levels of the substrate and / or metabolite of interest. In some embodiments, the kit includes reagents for measuring the levels of the substrate and / or metabolite of interest.

[0208] The components of the kit can be packaged either in the form of an aqueous medium or in lyophilized form. The container means of the kit generally includes at least one vial, test tube, flask, bottle, syringe or other container means into which the components can be placed and preferably can be suitably dispensed. If there are two or more components present in the kit, the kit also generally includes a second, third or other additional container into which additional components (e.g., a sterile pharmaceutically acceptable buffer and / or other diluent) can be separately placed. However, various combinations of the components may be included in one vial. The kits of the present invention also typically include means for containing nucleic acids and any other reagent containers tightly packed for commercial sale. Such containers include injection-molded or blow-molded plastic containers in which the desired vials are held.

[0209] This component of the kit is for administration as described above. It may also include a pharmaceutically acceptable carrier or vehicle. This can also be in the form of microparticles, for example, if the composition is in the form of tablets or powders. The term "carrier" refers to a diluent, adjuvant or excipient administered together with the substrate. Such pharmaceutical carriers include liquids such as water and oils, and oils include those derived from petroleum, animal, plant or synthetic sources, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc. The carrier can be physiological saline, gum arabic, gelatin, gelatinized starch, talc, keratin, colloidal silica, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants and coloring agents can also be used. Physiological saline solution, as well as aqueous solutions of dextrose and glycerol, can also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical carriers also include excipients, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol. The composition may also optionally contain a small amount of wetting agent or emulsifier, or pH buffer. The composition can be in the form of a liquid, for example, a solution, emulsion or suspension. The liquid can be useful for delivery by injection, infusion (e.g., IV infusion) or subcutaneous administration. As a solid composition for oral administration, the composition can be formulated into forms such as powders, granules, compressed tablets, pills, capsules, chewing gums, wafers, etc. The composition can take the form of one or more dosage units.

[0210] The substrate can be contained in a composition such as a nutritional supplement. Differences in the absorption rate of the substrate into the blood can cause a significant shift in the time of the maximum concentration in the exhaled breath. Thus, in one embodiment, the substrate is provided in the formulation to ensure rapid delivery. In one embodiment, the substrate is formulated as a liquid. In another embodiment, the substrate is formulated as a rapidly releasing / rapidly dissolving tablet or capsule. This ensures that the absorption has a time constant that is much shorter compared to washout.

[0211] In another embodiment, the subject has fasted overnight, and the fasting can be combined with the provision of a substrate as a liquid or a rapidly disintegrating / dissolving tablet or a rapidly disintegrating / dissolving capsule or other oral dosage form.

[0212] Typically, the amount of substrate administered as part of the method of the present invention, or the amount of substrate included in the composition included in the kit, is at least about 0.01% substrate relative to the weight of the composition. When intended for oral administration, this amount can vary in the range of about 0.1% to about 80% by weight of the composition. For administration by injection, the composition can typically include from about 0.1 mg / kg to about 250 mg / kg of the subject's body weight, preferably from about 0.1 mg / kg to about 20 mg / kg of the subject's body weight, more preferably from about 1 mg / kg to about 10 mg / kg of the subject's body weight.

[0213] Use The present invention also relates to the use of an exogenous substrate of an enzyme in any of the methods described herein. In one embodiment, the exogenous substrate is selected from nonanal, butanol, trans-2-hexenal, hexanal, benzaldehyde, citral and / or 2-pentanone.

[0214] The present invention also relates to nonanal, butanol, or one or more of their metabolites for use in a method of detecting, staging, monitoring, or predicting the prognosis of NASH in a subject, the method comprising the step of measuring the concentration of one or more of nonanal, butanol, or their metabolites in a biological sample obtained from the subject. The present invention also relates to nonanal, butanol, trans-2-hexenal, hexanal, benzaldehyde, citral, 2-pentanone, nonanol, butanone, trans-2-hexenol, hexanol, benzyl alcohol, nerol, 2-pentanol, benzoic acid, hippuric acid, 2,3-butanediol, 3-hydroxy-2-pentanone, and / or 2,3-pentanediol for use in an in vivo, in vitro, or ex vivo method of detecting, screening, monitoring, diagnosing, or predicting the prognosis of NASH in a subject.

[0215] One aspect of the present invention relates to the use of nonanal, butanol, trans-2-hexenal, hexanal, benzaldehyde, citral, 2-pentanone, nonanol, butanone, trans-2-hexenol, hexanol, benzyl alcohol, nerol, 2-pentanol, benzoic acid, hippuric acid, 2,3-butanediol, 3-hydroxy-2-pentanone and / or 2,3-pentanediol as biomarkers for NASH disease. Also within the scope of the present invention is the use of nonanal, butanol, trans-2-hexenal, hexanal, benzaldehyde, citral, 2-pentanone, nonanol, butanone, trans-2-hexenol, hexanol, benzyl alcohol, nerol, 2-pentanol, benzoic acid, hippuric acid, 2,3-butanediol, 3-hydroxy-2-pentanone and / or 2,3-pentanediol as a biomarker for one or more of NASH without fibrosis, NASH with fibrosis, or NASH-cirrhosis.

[0216] The present invention also relates to nonanal, butanol, trans-2-hexenal, hexanal, benzaldehyde, citral, 2-pentanone, nonanol, butanone, trans-2-hexenol, hexanol, benzyl alcohol, nerol, 2-pentanol, limonene, benzoic acid, hippuric acid, 2,3-butanediol, 3-hydroxy-2-pentanone, 2,3-pentanediol and / or perillyl alcohol for use in a method for distinguishing NAFL from NASH in a subject, the method comprising measuring the concentration of one or more of nonanal, butanol, trans-2-hexenal, hexanal, benzaldehyde, citral, 2-pentanone, nonanol, butanone, trans-2-hexenol, hexanol, benzyl alcohol, nerol, 2-pentanol, limonene, benzoic acid, hippuric acid, 2,3-butanediol, 3-hydroxy-2-pentanone, 2,3-pentanediol and / or perillyl alcohol in a biological sample obtained from the subject.

[0217] Unless otherwise defined in this specification, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by those skilled in the art. The foregoing disclosure provides a general description of the subject matter included within the scope of the present invention, including methods of making and using the invention and its best mode, but the following examples are provided to further enable those skilled in the art to practice the invention and to provide a complete written description thereof. However, those skilled in the art will understand that the details of these examples should not be read as limiting the invention, and that the scope of the invention should be understood from the claims appended to this disclosure and their equivalents. Various further aspects and embodiments of the invention will be apparent to those skilled in the art in view of this disclosure.

[0218] All documents mentioned in this specification are hereby incorporated by reference in their entirety.

[0219] "And / or" as used in this specification shall be construed as a specific disclosure of each of two specified features or components, either with or without the other. For example, "A and / or B" shall be construed as (i) A, (ii) B, and (iii) A and B, respectively, as if each were individually described herein. Except where otherwise specified in the context, the above descriptions and definitions of features are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments described.

[0220] The present invention will be further described by the following non-limiting examples.

Example

[0221] (Example 1) Identification of Genes Expressed with Differences in Comparison between NASH Tissue and Healthy / NAFLD Tissue The gene expression datasets were obtained from dedicated repositories. These datasets were generated from liver tissues obtained from healthy subjects or subjects suffering from NAFLD with disease stages ranging from simple steatosis to NASH and fibrosis. Through analysis of genes that are differentially expressed between subjects with NASH and those with simple steatosis or healthy subjects, AKR1B10 was identified as a gene upregulated in NASH at the transcriptome level. Further investigation of available literature showed that AKR1B10 also increases at the protein level in NASH.

[0222] (Example 2) Production of Biomass in Cell-Based Assays The substrates identified for AKR1B10 are nonanal, trans-2-hexenal, hexanal, benzaldehyde, and citral. The inventors examined the conversion of these substrates to their respective biomass products, 1-nonanol, trans-2-hexenol, hexenol, benzyl alcohol, and nerol, in cancer cell lines overexpressing AKR1B10. Addition of the substrates to the medium results in conversion to the biomass products within a 6-hour time frame. Removal of AKR1B10 or treatment of the cells with an AKR inhibitor results in a decrease or absence of biomass production.

[0223] A549 lung cancer cells were targeted with the CRISP / Cas system to ablate the protein expression of AKR1B10. An sgRNA specific to the AKR1B10 gene was co-transfected into the cells with SpCas9 using ribonucleoprotein (RNP). Clone cells were generated using limiting dilution. After growth, PCR was used to screen the clones, and a total of four correctly edited clones were identified. Protein extracts obtained from WT cells and AKR1B10 KO cells were analyzed using the Jess system, and the samples were probed with an AKR1B10-specific antibody and an antibody against α-tubulin as an independent reporter. Western blot analysis of cell extracts obtained from wild-type (WT) or AKR1B10 CRISP / Cas knockout (KO) revealed that four different clones were generated. The absence of antibody staining against AKR1B10 in the KO cell lysates indicates complete and constitutive removal of this enzyme. The results are shown in Figure 1.

[0224] 5×10 4 Individuals / mL of WT cells or 3 AKR1B10 KO clone cells were seeded in 24-well plates using cell culture medium, allowed to adhere, and grown for 48 hours until confluent. They were then treated with 30 μM nonanal or 10 μM trans-2-hexenal. Aliquots of the medium were collected 0.5, 1, and 3 hours after treatment. Headspace analysis was performed by combining the Centri and GC-MS Orbi-trap systems. Peak identification of nonanal, 1-nonanol, trans-2-hexenal, and trans-2-hexenol was performed by comparing the peaks to reference standards. The results are shown in Figure 2.

[0225] The medium incubated for 3 hours with the addition of 30 μM nonanal shows a decrease in nonanal due to natural evaporation. The addition of the medium with 30 μM nonanal to the WT cell line or AKR1B10 KO cell line shows an increase in the decrease over 3 hours, indicating that the cells are metabolizing nonanal (Figure 2A). Under all conditions, when only methanol is added to the medium, nonanal is not detected. In Figure 2B, the same medium as in Figure 2A with the addition of 30 μM nonanal was used. This shows that 1-nonanol is not produced in the absence of cells. The addition of the medium to WT cells or KO cells shows the production of 1-nonanol at a level three times higher in WT cells compared to KO cells. Under all conditions, when nonanal is not added to the medium, 1-nonanol is not detected.

[0226] The medium incubated for 3 hours with the addition of 10 μM trans-2-hexenal shows a decrease in trans-2-hexenal due to natural evaporation. The addition of the medium with 10 μM trans-2-hexenal to the WT cell line or AKR1B10 KO cell line shows an increase in the decrease over 3 hours, indicating that the cells are metabolizing trans-2-hexenal. Under all conditions, when only methanol is added to the medium, trans-2-hexenal is not detected. Figure 2D. The same medium used in Figure 2C with the addition of 10 μM trans-2-hexenal shows no production of trans-2-hexenol in the absence of cells. The addition of the medium to WT cells or KO cells shows the production of trans-2-hexenal at a level ten times higher in WT cells compared to KO cells. Under all conditions, when trans-2-hexenal is not added to the medium, trans-2-hexenol is not detected.

[0227] These results indicate that nonanal and trans-2-hexenal are substrates of AKR1B10 and that they are converted by this enzyme to 1-nonanol and trans-2-hexenol, respectively.

[0228] (Example 3) Washout Experiment of Nonanal and 2-Butanol The analysis of the washout of nonanal and 2-butanol in breath samples collected from three volunteers was conducted before and after the ingestion of 500 mg of nonanal and 100 mg of 2-butanol. Three breath collections were performed for each volunteer. The areas of nonanal and 2-butanol, as well as their respective bioproducts, nonanol and 2-butanone, in the collected breath samples showed intensity spikes within 20 minutes among the three volunteers.

[0229] Three volunteers participated in this test three times. They were made to swallow an emulsion containing 500 mg of nonanal and 100 mg of 2-butanol, and their breath was collected using a ReCIVA, a face mask containing a breath collection tube, and storage of breath VOCs at eight different time points before and after the ingestion of the emulsion.

[0230] The appearance of the probes in breath and their conversion to bioproducts were measured using gas chromatography-mass spectrometry (GC-MS).

[0231] Nonanal and 2-butanol were detected in the breath samples of the three volunteers collected after the ingestion of the emulsion. The level of nonanal increased within 20 minutes. Nonanol showed the same trend as nonanal, with a spike seen within 20 minutes after the ingestion of nonanal. The level of 2-butanol increased within 20 minutes. 2-Butanone showed the same trend as 2-butanol, with a spike seen within 20 minutes after the ingestion of 2-butanol.

[0232] The washout is shown in Figure 3.

[0233] (Example 4) Detection of VOCs from Healthy Human Hepatocytes Objective To examine the metabolism of 2-butanol and nonanal by healthy human hepatocytes over 24 hours and compare it with the change in the concentration of these compounds over the same period on a cell-free control plate.

[0234] Purpose · To understand the metabolism of 2-butanol and nonanal in primary hepatocytes. · To evaluate and compare the changes in the levels of these compounds in the medium containing hepatocytes over 24 hours from addition, compared with the medium without hepatocytes. · To evaluate the metabolic degradation rate of the designated VOCs by healthy hepatocytes.

[0235] Methods Organ-on-a chip samples were used.

[0236] Organ-on-a chip is a three-dimensional hepatocyte culture system developed by a company called CN-Bio Ltd. Aliquots of the medium collected at various time points after VOC administration are analyzed using CENTRI-GC-MS, a system for VOC headspace detection and quantification.

[0237] The experiments included samples from healthy hepatocytes in the medium exposed to 2-butanol and nonanal at one of the concentrations of 50, 10, 2, and 0 ng / μL, and a control, which was a medium without hepatocytes containing 2-butanol and nonanal at the same concentration.

[0238] System suitability tests (SST) and confirmation with two standards (0, 2, 10, and 50 ng / μL) were performed twice each at the beginning, middle, and end of the sequence to reduce the risk of instrument deviation.

[0239] To reduce the risk of carryover, tests were also performed on empty tubes at the beginning, middle, and end of the sequence, and 0.5 μL of an internal standard (1000 ng / μL) was added to each sample before analysis.

[0240] Conclusion Samples from plates containing only media with hepatocytes and media treated with 2-butanol and nonanal were analyzed. eVOCs of 50, 10, 2, and 0 ng / μL were used. When the instrument performance was confirmed, good consistency was shown throughout the sequence. 2-Butanol and nonanal showed a decrease in both media with only media and media with hepatocytes. When exposed to media with only hepatocytes and media with only 1-nonanol (a biological product of nonanal) and 2-butanone (a biological product of 2-butanol), metabolic rates were observed for all compounds, and spikes corresponding to the metabolism of nonanal and 2-butanol and subsequent decreases were shown. This is shown in Figures 4 and 5.

[0241] (Example 5) Detection of VOCs from healthy human hepatocytes and diseased human hepatocytes Organ-on-a-chip containing healthy human hepatocytes and human hepatocytes treated to create a model of healthy or NASH liver disease was used in the following experiments. The Organ-on-a-chip containing healthy human hepatocytes was the same as the above Organ-on-a-chip, but the experiments also included a model of NASH hepatocytes. The Organ-on-a-chip was as described above, but the experiments also included a model of NASH hepatocytes. The latter was obtained by treating healthy hepatocytes with Kupffer cells and hepatic stellate cells. These additional cells induce inflammation and fibrosis accumulation at levels similar to those observed in NASH liver. This model was verified by gene expression and protein expression and showed a profile consistent with that observed in NASH liver biopsied from diseased subjects.

[0242] As described in more detail below, healthy hepatocytes and NASH hepatocytes showed differential metabolism for nonanal and 2-butanol. NASH hepatocytes overexpressing AKR1B10 showed a higher production of 1-nonanol when treated with nonanal, compared to healthy hepatocytes. Conversely, NASH hepatocytes showed a decrease in the production of 2-butanone when treated with 2-butanol, which is as expected considering the downregulation of the alcohol dehydrogenase pathway reported in NASH.

[0243] Primary human hepatocytes (PHH), Kupffer cells (KC), and hepatic stellate cells (HSC) were seeded on a CN-Bio PhysioMimix LC12 MPS culture plate at 6×10 5 cells per well for PHH and 6×10 4 cells for HKC and HSC (required to induce the NASH phenotype) in CN-Bio seeding medium. Throughout the experiment, the cells were maintained at a medium flow rate of 1 μl / s. On days 4, 6, 8, 11, and 12 after seeding, medium aliquots were taken to measure LDH and albumin to assess the health status of the cells. On day 13 (24 hours after VOC treatment), the final medium was collected for analysis. The scaffolds were fixed in 4% formaldehyde and stained with Oil Red O to evaluate lipid accumulation.

[0244] Lactate dehydrogenase levels (LDH) were evaluated using the CytoTox 96 Cytotoxicity (LDH) Assay Kit. Albumin was measured using the AssayMax Albumin ELISA Kit. IL-6 and TIMP-1 ELISA (R&D DuoSet kits) were performed on medium samples collected on days 11 and 13 (24 hours after dosing) to evaluate the levels of inflammation and fibrosis. The results are shown in Figure 6.

[0245] When the levels of lactate dehydrogenase (LDH) were measured in the supernatants of healthy hepatocytes and NASH hepatocytes, it was shown that the levels decreased over the course of the culture days, indicating that there was no cell stress due to the culture procedure at the time when the cells were treated with nonanal and 2-butanol (6A). From the levels of albumin in the supernatants of hepatocytes, it was shown that albumin produced by NASH hepatocytes was less than that of healthy hepatocytes, which is as predicted considering the disease phenotype observed in humans (Figure 6B). Levels of the interleukin 6 (IL-6) and TIMP metallopeptidase inhibitor 1 (TIMP-1) markers of inflammation and fibrosis measured in the supernatants before and after treatment with nonanal and 2-butanol (Figures 6C and 6D).

[0246] To observe lipids, cultured hepatocytes were stained with Oil Red O to obtain microscopic images. As expected, NASH hepatocytes showed strong staining as shown in the microscopic images of Figure 7.

[0247] The data shown in Figures 6 and 7 indicate that hepatocyte culture was successfully performed and healthy and NASH phenotypes were generated.

[0248] Primary hepatocytes were treated with 2-butanol and nonanal diluted in hepatocyte medium to a concentration of 50 ng / μL. Aliquots of the medium were collected at 0.5, 1, 2, 4, 6, and 24-hour time points. The remaining medium-compound mixture not added to the cells was considered as the 0-hour reference. Headspace analysis was performed by combining Centri, an automated system used to collect VOCs in the headspace, with a GC-MS Orbitrap system, a high-resolution mass spectrometer for the detection and quantification of VOCs. Peak identification of 2-butanol, 2-butanone, nonanal, and 1-nonanol was performed by comparing the peaks with reference standards. A p-value < 0.05 was considered statistically significant.

[0249] Figure 8A. The level of 2-butanol decreases over time due to natural evaporation in the absence of hepatocytes. However, the presence of NASH hepatocytes and healthy hepatocytes shows a more significant decrease at the 24-hour time point. Figure 8B. The level of 2-butanone does not show an increase in the absence of hepatocytes. Healthy hepatocytes show a higher production of 2-butanone at the 6-hour time point, and the peak bottom area of healthy hepatocytes is larger than that of NASH. This is as predicted considering that the alcohol dehydrogenase pathway involved in the conversion from 2-butanol to 2-butanone is downregulated in NASH. 2 . Figure 8C.

[0250] Nonanal showed a decrease due to natural evaporation in the absence of hepatocytes. However, in the presence of hepatocytes, nonanal showed a more significant decrease. Figure 8D. Nonanol was not produced in the absence of hepatocytes, but NASH hepatocytes showed a higher production of nonanol compared to healthy hepatocytes (the peak area is larger for NASH than for healthy ones). This result is consistent with the overexpression of AKR1B10, an enzyme that converts nonanal observed in the liver of NASH to nonanol. 3 .

[0251] The inventors have shown for the first time that NASH-induced metabolic changes can be detected using exogenous volatile organic compound (EVOC) probes that are safe for human ingestion and appear in exhaled breath along with their bioproducts after administration. These data support the use of breath analysis using EVOC probes for induced volatolomics for NASH detection tests that can be used to diagnose / detect NASH, monitor the progression of NASH, or predict the prognosis of NASH.

[0252] (References) 1. Endo, S. et al. Kinetic studies of AKR1B10, human aldose reductase-like protein: endogenous substrates and inhibition by steroids. Arch Biochem Biophys 487, 1-9, doi:10.1016 / j.abb.2009.05.009 (2009). 2. Li, H., Toth, E. & Cherrington, N. J. Alcohol Metabolism in the Progression of Human Nonalcoholic Steatohepatitis. Toxicol Sci 164, 428-438, doi:10.1093 / toxsci / kfy106 (2018). 3. Kanno, M. et al. Serum aldo-keto reductase family 1 member B10 predicts advanced liver fibrosis and fatal complications of nonalcoholic steatohepatitis. J Gastroenterol 54, 549-557, doi:10.1007 / s00535-019-01551-3 (2019).

[0253] (Example 6) Detection of VOCs from NASH rat models Several groups of 3 Wistar Han rats were fed a normal diet (ND), and the same number of groups as the 3 rats were fed a choline-deficient high-fat diet (CDHFD), a treatment widely used to induce liver fibrosis in rodent models. 1When the liver fibrosis was evaluated after 8 weeks, as expected, the CDHFD rats were found to have a higher degree of fibrosis compared to the ND rats (Figures 9A, 9B, and 9C). The weight gain shown by the CDHFD rats was less compared to the ND rats (Figure 10). Ten weeks after the start of feeding, the rats were orally administered either benzyl alcohol (208 mg / Kg) or 2-butanol (440 mg / Kg) or 2-pentanone (160 mg / Kg), and blood samples (200 μl) were collected at the time points of 5, 15, 30 minutes, 1, 1.5, 2, 4, 8, 12, 24 hours before and after the administration.

[0254] The blood samples were analyzed using gas chromatography-mass spectrometry (GC / -MS) or HPLC to measure the metabolic bioproducts benzoic acid, 3-hydroxy-2-butanone, 2,3-butanediol, and 2,3-pentanediol.

[0255] Benzoic acid is a bioproduct of the alcohol dehydrogenase pathway and is further metabolized to hippuric acid. Chronic liver damage induces a decrease in hippuric acid metabolism. 2 In line with these reports, the inventors observed that after benzyl alcohol administration, the concentration of benzoic acid in the blood of CDHFD rats increased compared to ND rats. In particular, benzoic acid was not present before administration. The levels in the blood increased between 5 and 240 minutes after administration, and higher concentrations were found in CDHFD rats. The blood concentrations reached undetectable levels in all rats after 480 minutes (Figure 11).

[0256] 2-Butanol is converted to 2-butanone by alcohol dehydrogenase. Subsequently, 2-butanone is converted to 3-hydroxy-2-butanone and 2,3-butanediol by CYP enzymes. 3,4 The inventors found that after administering 2-butanol, the concentrations of 3-hydroxy-2-butanone and 2,3-butanediol increased 240 minutes after the administration. At 8 and 12 hours (480, 720 minutes) after the administration, these compounds showed higher levels in CDHFD compared to ND rats (Figures 12 and 13).

[0257] 2-Pentanone is converted to 2,3-pentanediol by CYP 4 . After administration of 2-pentanone, the blood level of 2,3-pentanediol increased after 1 hour, and the level was higher in CDHFD rats than in ND rats 4 hours later (Figure 14).

[0258] These data indicate that NASH induces liver changes that alter the metabolism of the test compound. These changes can be used for diagnostic purposes.

[0259] Materials and Methods All bioanalytical experiments were performed by WuXi AppTec (WuXi AppTec (Nantong) Co., Ltd. Address: 699 South Huashi Road, Qidong, Natong, Jiangsu, P.R. China. Telephone: 0513 - 83395562).

[0260] A total of 18 Wistar Han rats were acclimatized for 6 days. Thereafter, 9 rats were kept on normal diet, and 9 rats were switched to a choline-deficient high-fat diet (CDHFD) (L-amino acid diet containing 45 kcal% fat and 0.1% methionine, without added choline, and 1% cholesterol) (Research Diets, catalog number A16092003, https: / / researchdiets.com / en / formulas / a16092003). Unless otherwise specified, rats were always allowed free access to food and water. Body weight was measured weekly.

[0261] Eight weeks after the start of feeding, the rats underwent liver biopsy under survival conditions. The rats were deeply anesthetized with 3% isoflurane, and liver pieces from the left lobe were collected through a longitudinal incision (3 - 4 cm) on the abdominal skin slightly to the left side of the rat, just caudal to the xiphoid process. At the end of the surgery day, the rats were subcutaneously administered with tridin (2 mg / kg) and penicillin (10 w U / rat), and subsequently administered once a day for 2 - 3 days as needed, and monitored until fully recovered. The collected liver tissues were sliced, stained with hematoxylin / eosin, and the fibrotic tissues were microscopically quantified as a percentage of the total area. The rats were allowed to recover from the biopsy for 2 weeks before further treatment.

[0262] After 10 weeks of feeding treatment, in order to administer various compounds, the rats were randomly assigned to each group composed of 3 healthy rats and 3 NASH rats.

[0263] Each group received administration of an emulsion containing the compounds reported in Table 1 (Table 1) in the indicated amounts.

[0264]

Table 1

[0265] To deliver a target volume of 5 mL / kg body weight, each compound preparation was administered as an emulsion containing 15% Tween80 diluted with water. After the rats were fasted for 16 hours, the emulsion was orally administered by gavage.

[0266] Blood samples were collected before (0 minutes) and at the following time points after the administration of the compound: 5 minutes, 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours.

[0267] At each time point, approximately 200 μL of blood sample was collected into an EDTA-K2 tube and placed on wet ice until centrifuged. Plasma was generated from the blood sample by centrifugation at approximately 4°C and 3,200 g for 5 minutes.

[0268] A total of 100 μL of plasma was collected and transferred to Eppendorf tubes respectively.

[0269] Among these, 400 μL of internal standard in acetonitrile was added to 95 μL of plasma, vortexed for 30 seconds, and then centrifuged at 3220 g for 15 minutes. Unless otherwise specified, a total of 100 μL of the supernatant was used for the quantification of the compound.

[0270] The quantification of the target compound was performed as shown in Table 2.

[0271]

Table 2

[0272] The details of the analysis method are as follows: · 3-Hydroxy-2-butanone (internal standard = acetone): · 2,3-Butanediol and 2,3-pentanediol (internal standard = 1-heptanol)

[0273]

Table 3

[0274] · Benzoic acid (internal standard = hexanoic acid): Injected directly into the instrument.

[0275]

Table 4

[0276] The response of each compound was normalized by the response of the internal standard, and absolute quantification was performed against the calibration curve.

[0277] (References) 1. Nevzorova YA, Boyer-Diaz Z, Cubero FJ, Gracia-Sancho J. Animal models for liver disease - A practical approach for translational research. J Hepatol 2020;73(2):423-440. doi: 10.1016 / j.jhep.2020.04.011. PMID: 32330604 2. Saltzman A, Caraway WT. Cinnamic acid as a test substance in the evaluation of liver function. J Clin Invest 1953;32(8):711-719. doi: 10.1172 / JCI102785. PMID: 13069619 3. Morey TE, Booth M, Wasdo S, Wishin J, Quinn B, Gonzalez D, et al. Oral adherence monitoring using a breath test to supplement highly active antiretroviral therapy. AIDS Behav 2013;17(1):298-306. doi: 10.1007 / s10461-012-0318-7. PMID: 23001413 4. Dietz FK, Rodriguez-Giaxola M, Traiger GJ, Stella VJ, Himmelstein KJ. Pharmacokinetics of 2-butanol and its metabolites in the rat. J Pharmacokinet Biopharm 1981;9(5):553-576. doi: 10.1007 / BF01061026. PMID: 7334459

Claims

1. A method for detecting, staging, monitoring, or predicting the prognosis of non-alcoholic steatohepatitis (NASH) in a subject, comprising the steps of measuring the concentration of an exogenous substrate of an enzyme in a biological sample obtained from the subject, and / or measuring the concentration of a metabolite of the substrate, wherein the substrate is a generally recognized safe (GRAS) compound, and the enzyme is aldo-ketereductase (AKR), alcohol dehydrogenase, cytochrome P450 (CYP), aldehyde dehydrogenase, or glycine N-acyltransferase.

2. The method according to claim 1, further comprising the step of determining the stage of NASH, wherein the stage of NASH is selected from NASH without fibrosis, NASH with fibrosis, NASH with hepatocellular carcinoma (HCC), or NASH with cirrhosis such as decompensated cirrhosis.

3. The method according to claim 1 or 2, for detecting or predicting the prognosis of NASH with fibrosis.

4. The method according to any one of claims 1 to 3, wherein the enzyme is aldo-ketoreductase (AKR), optionally the AKR is a member of the AKR family 1, and optionally the AKR is AKR1B10.

5. The method according to any one of claims 1 to 4, wherein the substrate is selected from aldehydes and / or alcohols.

6. The method according to any one of claims 1 to 5, wherein the substrate is nonanal and / or the metabolite is nonanol, and optionally the substrate is 1-nonanal and / or the metabolite is 1-nonanol.

7. The method according to any one of claims 1 to 6, wherein the substrate is trans-2-hexenal and / or the metabolite is trans-2-hexanol, or the substrate is hexanal and / or the metabolite is hexanol, or the substrate is benzaldehyde and / or the metabolite is benzyl alcohol, or the substrate is citral and / or the metabolite is nerol.

8. The method according to any one of claims 1 to 3, wherein the enzyme is alcohol dehydrogenase.

9. The method according to claim 8, wherein the substrate is butanol and / or the metabolite is butanone, and / or the substrate is 2-butanol and / or the metabolite is 2-butanone, and / or the substrate is 2-pentanone, and / or the substrate is 2-pentanone and / or the metabolite is 2-pentanol, 3-hydroxy-2-pentanone or 2,3-pentanediol, and / or the substrate is benzyl alcohol and the metabolite is benzylaldehyde and / or benzoic acid.

10. The method according to claim 8, wherein the enzyme is an aldehyde dehydrogenase, and optionally the substrate is benzaldehyde and the metabolite is benzoic acid.

11. The method according to any one of claims 1 to 3, wherein the enzyme is cytochrome P450 (CYP), and optionally the CYP is CYP1A1, CYP1A2, CYP1B1, CYP2, CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP2W1, CYP3, CYP3A4, CYP3A5, CYP3A7, or CYP3A43.

12. The method according to claim 11, wherein the substrate is 2-butanone and the metabolite is 3-hydroxy-2-butanone and / or 2,3-butanediol, or the substrate is 2-pentanone and the metabolite is 2,3-pentanediol.

13. The method according to any one of claims 1 to 12, wherein the substrate is labeled, and optionally the label is 12C, 13C, 14C, 2H, 14N, or 18O, or the substrate is not labeled.

14. The method according to any one of claims 1 to 13, wherein the biological sample is selected from breath, urine, blood, serum, and / or tissue.

15. The method according to any one of claims 1 to 14, comprising the step of establishing a value for a test subject based on the concentration of the substrate or metabolite or combination thereof in the test subject, optionally comparing the value of the test subject with one or more reference values, and indicating the possibility of NASH by the difference between the value of the test subject and the reference values, optionally the reference value being a value for a subject diagnosed with NASH, or non-alcoholic fatty liver disease (NAFLD), or NASH progressing to decompensated cirrhosis or HCC, or the reference value being a value for a healthy subject.

16. The method according to any one of claims 1 to 15, wherein the concentrations of two or more exogenous substrates of the enzyme and / or the concentrations of two or more metabolites are measured, and optionally the subject is administered the exogenous substrates of the enzyme, and optionally the concentration of the metabolites is measured.

17. A method for determining the effectiveness of a treatment, comprising the step of evaluating the activity of an enzyme in a subject diagnosed with NASH by measuring the concentration of the exogenous substrate of the enzyme and / or the concentration of a metabolite of the substrate in a biological sample obtained from the subject, wherein the subject is being treated for NASH and the enzyme is aldo-ketoreductase (AKR), alcohol dehydrogenase, or cytochrome P450 (CYP).

18. The method according to claim 17, comprising the steps of analyzing a first biological sample obtained from the subject at a first time point, and subsequently analyzing one or more additional biological samples obtained from the subject at one or more additional time points, or their ratios, wherein the treatment for NASH is optionally a drug-based treatment comprising gastric bypass surgery and / or administration of at least one drug selected from statins, incretin analogs, metformin, rimonabant, thiazolidinediones, and orlistat.

19. A method for monitoring the progression of NASH in a subject, comprising the steps of measuring the concentration of an exogenous substrate of an enzyme in a biological sample obtained from the subject, and / or measuring the concentration of a metabolite of the substrate, wherein the substrate is a generally recognized safe (GRAS) compound, and the enzyme is aldo-ketoreductase (AKR), alcohol dehydrogenase, or cytochrome P450 (CYP).

20. A kit for the detection or prognosis prediction of NASH, comprising an enzyme substrate and / or a metabolite of the substrate, and a device for capturing a biological sample from a patient, wherein the substrate and / or metabolite is optionally selected from nonanal, butanol, trans-2-hexenal, hexanal, benzaldehyde, citral, nonanone, butanone, trans-2-hexenol, hexenol, benzyl alcohol, nerol, 3-hydroxy-2-butanone, 2,3-butanediol, benzoic acid, hippuric acid, 2-pentanone, and 2,3-pentanediol.

21. Use of an exogenous substrate and / or metabolite of an enzyme whose activity or expression is upregulated or downregulated in NASH, in a method for detecting or predicting the prognosis of NASH, wherein the substrate is selected from nonanal, butanol, trans-2-hexenal, hexanal, benzaldehyde, citral, benzoic acid, butanone, and 2-pentanone, and the metabolite is selected from nonanol, butanone, trans-2-hexenol, hexanol, benzyl alcohol, nerol, 2-pentanone, 3-hydroxy-2-butanone, 2,3-butanediol, hippuric acid, and 2,3-pentanediol.

22. Use of nonanal, butanol, trans-2-hexenal, hexanal, benzaldehyde, citral, 2-pentanone, nonanol, butanone, trans-2-hexenol, hexanol, benzyl alcohol, nerol, 2-pentanol, benzoic acid, hippuric acid, 2,3-butanediol, 3-hydroxy-2-pentanone and / or 2,3-pentanediol as biomarkers for NASH.

23. A method for distinguishing between NASH and other stages of NAFLD in a subject, comprising the steps of measuring the concentration of an exogenous substrate of an enzyme in a biological sample obtained from the subject, and / or measuring the concentration of a metabolite of the substrate, wherein the substrate is a generally recognized safe (GRAS) compound, optionally the activity or expression of the enzyme is upregulated or downregulated in NASH, and optionally the enzyme is aldo-ketereductase (AKR), alcohol dehydrogenase, or cytochrome P450 (CYP).

24. A method for detecting or predicting the prognosis of early non-alcoholic steatohepatitis (NASH) in a subject, comprising the steps of measuring the concentration of an exogenous substrate of an enzyme in a biological sample obtained from the subject, and / or measuring the concentration of a metabolite of the substrate, wherein the substrate is a generally recognized safe (GRAS) compound, and optionally the enzyme is aldo-ketereductase (AKR), alcohol dehydrogenase, or cytochrome P450 (CYP).

25. A method for determining the stage of NASH in a subject, comprising the steps of measuring the concentration of an exogenous enzyme substrate in a biological sample obtained from the subject, and / or measuring the concentration of a metabolite of the substrate, wherein the substrate is a generally recognized safe (GRAS) compound.

26. The method according to any one of claims 23 to 25, wherein the activity or expression of the enzyme is upregulated or downregulated in NASH, and optionally the enzyme is aldo-ketoreductase (AKR), alcohol dehydrogenase, or cytochrome P450 (CYP).

27. A method for detecting or predicting the prognosis of NASH in a subject, for detecting or predicting the prognosis of early NASH in a subject, for determining the stage of NASH in a subject, or for distinguishing NASH from other stages of NAFLD in a subject, comprising the steps of measuring the concentration of an exogenous substrate of an enzyme in a biological sample obtained from the subject, and / or measuring the concentration of a metabolite of the substrate, wherein the substrate is a generally recognized safe (GRAS) compound and the enzyme is not a CYP enzyme.

28. A method for detecting or predicting the prognosis of NASH in a subject, for detecting or predicting the prognosis of early NASH in a subject, for determining the stage of NASH in a subject, or for distinguishing NASH from other stages of NAFLD in a subject, comprising the steps of measuring the concentration of an exogenous substrate of an enzyme in a biological sample obtained from the subject, and / or measuring the concentration of a metabolite of the substrate, wherein the substrate is a generally recognized safe (GRAS) compound and the substrate is not limonene.

29. A method for determining the effectiveness of a treatment, comprising the step of evaluating the activity of an enzyme in a subject diagnosed with NASH by measuring the concentration of the exogenous substrate of the enzyme and / or the concentration of a metabolite of the substrate in a biological sample obtained from the subject, wherein the enzyme is not a CYP enzyme or the substrate is not limonene.

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