Detection of liver disease

Abstract

The present invention relates to methods of detecting, staging, monitoring or prognosing non-alcoholic steatohepatitis (NASH) in a subject. The method comprises 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. The substrates are generally believed to be safe (GRAS) compounds.

Description

Technical Field

[0001] The present invention relates to a detection method for non-alcoholic steatohepatitis (NASH) and related methods and kits. Background Art

[0002] Nonalcoholic fatty liver disease (NAFLD) covers the full histological spectrum from simple benign liver steatosis to nonalcoholic steatohepatitis (NASH), which is characterized by lipid accumulation, inflammation, hepatocellular ballooning, and varying degrees of fibrosis. NASH can progress to cirrhosis or hepatocellular carcinoma (HCC). NASH, unlike non-alcoholic fatty liver (NAFL), has the greatest potential to progress to cirrhosis, liver failure, and liver cancer. The prevalence of NAFLD is increasing and is associated with an increase in obesity. Although knowledge of obesity-related liver disease is growing, the pathogenesis 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. Differentiating NASH from simple steatosis is very important for the clinical management of NAFLD patients and reducing mortality (Chen et al. Radiology. 2011 Jun; 259(3):749-756).

[0004] The only test approved for the diagnosis of NASH is a liver biopsy, an invasive procedure that can result in complications. Alternative methods lack adequate performance early and overall, which limits early detection of NASH and makes it difficult to assess the efficacy of experimental drugs. Therefore, alternative diagnostic tests are needed, particularly tests that can diagnose NASH and differentiate NASH from NAFL, another stage of NAFLD. Noninvasive differentiation of NAFLD and NASH will help significantly reduce the risks associated with the diagnosis of these pathologies. Differentiating NASH from NAFL will lead to earlier lifestyle changes, medical interventions, cancer screening, and overall improved outcomes.

[0005] The use of exogenous volatile organic compound (EVOC®) probes for induced volatomics - monitoring the metabolic processes of exogenous compounds by monitoring exhaled breath - to detect liver disease has been described in WO2019220145. The use of the probes described herein for the detection, diagnosis, staging, monitoring and prognosis of NASH provides an alternative test method for NASH. Summary of the invention

[0006] The inventors have demonstrated that NASH-induced metabolic changes are detectable using EVOC probes that are safe for human consumption and appear in the breath along with their bioproducts after administration. These data suggest that breath analysis of induced volatileomics using EVOC probes can be used for NASH detection testing, as well as screening, diagnosis, staging, monitoring, and prognosis.

[0007] In this method, one or more target compounds that are present in the breath exhaled by the subject and metabolized by the liver are measured in the breath gas after administration. The amount of one or more compounds (compound(s)) in the breath of the subject depends on the efficiency of the liver to clear the compound after administration. Therefore, the breath amount is a proxy for liver function. Volatile bioproducts can be detected alternatively or additionally to improve the accuracy of the diagnosis. Therefore, the amount of the compound in the breath can be used as an indicator of liver function affected in NASH.

[0008] Therefore, the present inventors have developed a non-invasive test for detecting and staging non-alcoholic fatty liver disease (NAFLD) that has progressed to non-alcoholic steatohepatitis (NASH) stage for detection, diagnosis, staging, monitoring and prognosis in subjects who have or are suspected of having liver disease (i.e., at risk of progression to more advanced liver disease stages).

[0009] Therefore, in a first aspect, the present invention relates to a method for detecting or prognosing NASH 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, wherein 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.

[0010] The method further comprises 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.

[0011] In one embodiment, the enzyme is aldo-keto reductase (AKR).

[0012] In one embodiment, the AKR is an AKR family 1 member.

[0013] In one embodiment, the AKR is AKR1B10. In one embodiment, the substrate is selected from aldehydes and / or alcohols.

[0014] In one embodiment, the substrate is nonanal and / or the metabolite is nonanol.

[0015] In one embodiment, the substrate is 1-nonanal and / or the metabolite is 1-nonanol.

[0016] In one embodiment, the substrate is trans-2-hexenal and / or the metabolite is trans-2-hexanol.

[0017] In one embodiment, wherein the substrate is hexanal and / or the metabolite is hexanol.

[0018] In one embodiment, the substrate is benzaldehyde and / or the metabolite is benzyl alcohol.

[0019] In one embodiment, the substrate is citral and / or the metabolite is nerol.

[0020] In one embodiment, the enzyme is an alcohol dehydrogenase.

[0021] In one embodiment, the substrate is butanol and / or the metabolite is butanone.

[0022] In one embodiment, the substrate is 2-butanol and / or the metabolite is 2-butanone.

[0023] In one embodiment, the substrate is 2-pentanone.

[0024] In one embodiment, the substrate is 2-pentanone and / or the metabolite is 2-pentanol, 3-hydroxy-2-pentanone or 2,3-pentanediol.

[0025] In one embodiment, the substrate is benzyl alcohol and the metabolite is benzaldehyde and / or benzoic acid.

[0026] In one embodiment, the enzyme is aldehyde dehydrogenase.

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

[0028] In one embodiment, the enzyme is cytochrome P450 (CYP).

[0029] In one embodiment, the CYP is CYP1A1, CYP1A2, CYP1 B1, 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.

[0030] In one embodiment, the substrate is 2-butanone and the metabolite is 3-hydroxy-2-butanone and / or 2,3-butanediol.

[0031] In one embodiment, the substrate is 2-pentanone and the metabolite is 2,3-pentanediol.

[0032] In one embodiment, the enzyme is glycine N-acyltransferase.

[0033] In one embodiment, the substrate is benzoic acid and the metabolite is hippuric acid.

[0034] In one embodiment, the substrate is labeled, for example, with 12C, 13C, 14C, 2H, 14N or 18O.

[0035] In one embodiment, the substrate is unlabeled.

[0036] In one embodiment, the biological sample is selected from respiratory gas, urine, blood, serum and / or tissue.

[0037] In one embodiment, the method comprises establishing a test subject value based on the concentration of the substrate or metabolite in the test subject.

[0038] In one embodiment, the test subject value is compared to one or more reference values, wherein a difference between the test subject value and the reference value indicates the likelihood of NASH.

[0039] In one embodiment, the reference value is a value for a subject who has been diagnosed as having NASH.

[0040] In one embodiment, the reference value is a value for a subject who has been diagnosed with non-alcoholic fatty liver disease (NAFLD).

[0041] In one embodiment, the reference value is a value for a NASH subject with cirrhosis and / or HCC that has progressed to a decompensated stage.

[0042] In one embodiment, the reference value is a value for healthy subjects.

[0043] In one embodiment, the concentrations of two or more exogenous substrates and / or the concentrations of two or more metabolites of the enzyme are measured.

[0044] In one embodiment, the subject has been administered an exogenous substrate for the enzyme.

[0045] In one embodiment, the concentration of a metabolite is measured.

[0046] In another aspect, the present invention relates to a method for determining the effect of a treatment, comprising assessing the activity of an enzyme in a subject diagnosed with NASH 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 subject has been treated for NASH, and wherein the enzyme is aldo-keto reductase (AKR) or alcohol dehydrogenase or cytochrome P450 (CYP).

[0047] In one embodiment, the method comprises analyzing a first biological sample obtained from said subject at a first time point, and then analyzing one or more additional biological samples obtained from said subject at one or more additional time points, or a ratio thereof.

[0048] In one embodiment, the treatment of NASH is gastric bypass surgery and / or a drug-based treatment comprising the administration of at least one drug.

[0049] In one embodiment, the treatment of NASH is gastric bypass surgery and / or a drug-based therapy comprising the administration of at least one drug selected from the group consisting of statins, incretin mimetics, metformin, rimonabant, thiazolidinediones, and orlistat.

[0050] In another aspect, the invention relates to a method for monitoring the progression or regression of NASH 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 wherein the enzyme is an aldo-keto reductase (AKR) or an alcohol dehydrogenase or a cytochrome P450 (CYP).

[0051] In another aspect, the present invention relates to a kit for the detection or prognosis of NASH, the kit comprising a substrate for an enzyme and / or a metabolite of the substrate and a device for capturing a biological sample from a patient.

[0052] 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.

[0053] In another aspect, the present invention relates to the use of exogenous substrates and / or metabolites of an enzyme in a method for detecting or prognosing NASH, wherein the activity or expression of the enzyme is upregulated or downregulated in 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.

[0054] In another aspect, the present invention relates to a method 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 for detecting or prognosing NASH in a subject in vivo, 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.

[0055] 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 NASH detection, staging (i.e., assessing NASH stage), diagnosis, monitoring and progression.

[0056] In another aspect, the present invention relates to a method of distinguishing 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.

[0057] In one embodiment, the activity or expression of the enzyme is upregulated or downregulated in NASH.

[0058] In one embodiment, the enzyme is an aldo-keto reductase (AKR) or an alcohol dehydrogenase or a cytochrome P450 (CYP).

[0059] In another aspect, the present invention relates to a method for detecting or prognosing early non-alcoholic steatohepatitis (NASH) 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. In one embodiment, the activity or expression of the enzyme is upregulated or downregulated in NASH. In one embodiment, the enzyme is an aldo-keto reductase (AKR) or an alcohol dehydrogenase or a cytochrome P450 (CYP).

[0060] In another aspect, the present invention relates to a method for determining the stage of NASH 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. In one embodiment, the activity or expression of the enzyme is upregulated or downregulated in NASH.

[0061] In one embodiment, the enzyme is an aldo-keto reductase (AKR) or an alcohol dehydrogenase or a cytochrome P450 (CYP).

[0062] In another aspect of the invention, the present invention relates to a method for determining the stage of NASH 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.

[0063] In one embodiment, the activity or expression of the enzyme is upregulated or downregulated in NASH.

[0064] In one embodiment, the enzyme is an aldo-keto reductase (AKR) or an alcohol dehydrogenase or a cytochrome P450 (CYP).

[0065] Therefore, in another aspect, the present invention relates to a method for detecting or prognosing NASH in a subject, detecting or prognosing early NASH in a subject, determining the stage of NASH in a subject, monitoring the progression or regression of NASH in a subject, or distinguishing NASH from other stages of NAFLD in a subject, 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, wherein the enzyme is not a CYP enzyme.

[0066] In another aspect, the present invention relates to a method for detecting or prognosing NASH in a subject, detecting or prognosing early NASH in a subject, determining the stage of NASH in a subject, monitoring the progression or regression of NASH in a subject, or distinguishing NASH from other stages of NAFLD in a subject, 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, wherein the substrate is not limonene.

[0067] In another aspect, the present invention relates to a method for determining the effect of a treatment, comprising assessing the activity of an enzyme in a subject diagnosed with NASH 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 wherein the substrate is not limonene.

[0068] In all of the above aspects, combinations of substrates and / or metabolites may be used as further explained below. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 Western blot analysis of cell extracts obtained from wild-type (WT) or AKR1B10 CRISP / Cas knockout (KO).

[0070] Figure 2 .Headspace analysis of WT and KO cells. 2A and B: medium supplemented with 30 µM nonanal; 2C and D: medium supplemented with 10 µM trans-2-hexenal.

[0071] Figure 3 .Washouts from healthy volunteers.

[0072] Figure 4 .Detection of nonanal and nonanol in healthy human hepatocytes.

[0073] Figure 5 . Detection of butanol and butanone in healthy human hepatocytes.

[0074] Figure 6 A. Lactate dehydrogenase (LDH) levels measured in supernatants of healthy and NASH hepatocytes. 6B. Serum albumin measured in supernatants of healthy and NASH hepatocytes. 6C. Interleukin 6 (IL-6) levels. 6D. TIMP metallopeptidase inhibitor 1 (TIMP-1) levels.

[0075] Figure 7 .Microscope images of cultured hepatocytes.

[0076] Figure 8.Analysis of NASH and healthy liver cells. 8A. Levels of 2-butanol. 8B. Levels of 2-butanone. 8C. Levels of nonanal. 8D. Levels of nonanol.

[0077] Fig. 9 A) NASH rat model timeline showing rat age and actions taken at each time point. B) Graph showing the number of Wistar Han rats fed a normal diet and a choline-deficient high-fat diet. C) Box plot of fibrosis area according to disease status, showing that rats fed a choline-deficient high-fat diet exhibited symptoms of NASH.

[0078] Fig.10 .Box plots of rat body weight according to days after disease and diet initiation.

[0079] Fig.11 .Box plots of benzoic acid, by time and disease.

[0080] Fig.12 . Box plots of 3-hydroxy-2-butanone according to time (minutes) and disease.

[0081] Fig.13 . Box plots of 2,3-butanediol according to time (minutes) and disease.

[0082] Fig.14 . Box plots of 2,3-pentanediol according to time (minutes) and disease. DETAILED DESCRIPTION OF THE INVENTION

[0084] The invention will now be further described. In the following paragraphs, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects, unless there is a clear indication to the contrary. In particular, any feature specified as being preferred or advantageous may be combined with any other feature specified as being preferred or advantageous.

[0085] method

[0086] The present invention provides methods for detecting, staging, screening, diagnosing, monitoring or prognosing NAFLD that has progressed to the NASH stage. Advantageously, the inventors have developed a method for detecting NASH that can be non-invasive or minimally invasive because the method is performed on a biological sample. In addition, the inventors have also developed a method for identifying the stage of the disease, i.e., how far the disease has progressed, and for distinguishing between NASH and NAFL.

[0087] In a first aspect, the present invention relates to a method for detecting or prognosing NAFLD that progresses to NASH stage in a subject, the method comprising measuring the concentration of an exogenous substrate of a 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 a generally recognized as safe (GRAS) compound, wherein the enzyme is aldo-keto reductase (AKR) or alcohol dehydrogenase or cytochrome P450 (CYP) or aldehyde dehydrogenase or glycine N-acyltransferase.

[0088] NAFLD is a term that refers to a range of conditions caused by fat accumulation within the liver. The term covers a spectrum of diseases that includes a mild, benign form of the disease in which fat accumulation is present in the liver, called steatosis or NAFL. This disease state can progress to a more severe form called NASH, in which the liver becomes inflamed, and this stage may also be referred to herein as NASH without fibrosis. NASH can then progress to NASH with fibrosis, in which ongoing inflammation causes scar tissue to form around the liver and nearby blood vessels. The most severe form of this disease is NASH with cirrhosis, which can occur after long-term inflammation, causing the liver to shrink and scar. NASH with cirrhosis causes permanent damage to the liver and can lead to liver failure and the development of liver cancer, hepatocellular carcinoma (HCC).

[0089] 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 the liver functions normally. There are usually no symptoms, and patients may not realize they have the condition. Although fat deposits are considered harmless, it is important to prevent the disease from progressing to the next stage.

[0090] Stage 2 is often referred to as NASH. NASH is a more aggressive form of the condition in which the liver has become inflamed. Inflammation is the body's healing response to injury or damage, and in this case, is a sign that liver cells have become damaged. Patients with NASH may have a dull or aching pain in the upper right side of their abdomen (below right side of their rib cage). NASH may occur with or without fibrosis.

[0091] Stage 3 is often characterized by cirrhosis. In this most severe stage, bands of scar tissue and clumps of liver cells form. The liver shrinks and forms lumps, which is called cirrhosis. Cirrhosis progresses slowly, gradually causing the liver to stop functioning. The damage caused by cirrhosis is irreversible, and people may develop signs of liver failure. Cirrhosis tends to develop after age 50, usually after years of liver inflammation associated with earlier stages of the disease. People with cirrhosis caused by NAFLD often also have type 2 diabetes.

[0092] In an embodiment, the method further comprises determining the stage of NASH, wherein the stage of NASH is selected from: NASH without fibrosis (which will progress to fibrosis) or NASH with fibrosis. In some embodiments, NASH induced by cirrhosis, such as decompensated cirrhosis or hepatocellular carcinoma (HCC) is also within the scope of the present invention.

[0093] The terms "cirrhosis," "liver cirrhosis," or "hepatic cirrhosis" refer to a condition in which the liver no longer works properly because of long-term damage. This damage is characterized by the replacement of normal liver tissue with scar tissue, called fibrosis. The disease usually progresses slowly over months or years and often has no symptoms. Eventually, excessive scarring leads to a loss of liver function.

[0094] The term "prognosis" refers to the prediction or likely outcome of a disease. As used herein, it refers to the likely outcome of a liver disease, including whether the disease (e.g., NASH) will respond to treatment or palliative measures and / or the likelihood of disease progression.

[0095] The term progression as used herein may refer to the progression of a disease state. The term regression as used herein refers to a decrease in the severity of a disease state. When monitoring a disease, this may result in detecting progression or regression.

[0096] Remission may be achieved as a result of changes in health patterns or therapeutic interventions, such as using the treatments described herein, including treatments in clinical drug trials.

[0097] In one embodiment, the stage is early NASH. Early NASH is distinct from NAFL and is characterized by steatosis, inflammation, high liver fat, and hepatocellular damage.

[0098] The method of the present invention is based on administering an exogenous substrate to a subject. An "exogenous substrate" is any compound that can be administered to a subject that is metabolized by an enzyme in the subject. An exogenous substrate refers to a compound that is recognized by a target enzyme, and the enzyme catalyzes the conversion of the substrate into a different compound referred to herein as a "metabolite". The substrate used in the method of the present invention is an exogenous substance, i.e., a xenobiotic substance. The term xenobiotic substance refers to a substance that is foreign to the subject's body that 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 substance that is not normally present in the subject's body. In an embodiment, the exogenous substrate is a generally recognized as safe (GRAS) compound.

[0099] The exogenous substrate is selectively metabolized by an enzyme in the subject. The enzyme is an enzyme whose activity or expression is downregulated in NASH or whose activity or expression is upregulated in NASH, i.e., in patients with NASH disease, compared to healthy subjects. For example, the activity or expression in liver tissue of NASH patients is upregulated or downregulated compared to healthy subjects. Therefore, changes in the expression and / or activity of the enzyme are indicative of NASH.

[0100] For example, a gene encoding an enzyme may be differentially expressed in NASH tissues compared to non-NASH tissues. For example, the enzyme may be expressed at a higher level in NASH tissues than in non-NASH tissues, or at a lower level in NASH tissues than in non-NASH tissues. In another embodiment, the enzyme may have different activities in NASH tissues compared to non-NASH tissues. For example, the enzyme may be modified so that the activity of the enzyme in NASH tissues is higher or lower than that in non-NASH tissues. Gene expression can be measured by techniques known in the art, such as by mRNA quantification or by measuring cDNA. The activity of an enzyme can be measured by assessing its metabolic activity, i.e., the ability of the enzyme to metabolize a substrate.

[0101] Non-NASH tissue refers to, for example, healthy tissue or NAFLD tissue that has not progressed to NASH, such as tissue from a subject with NAFL also known as steatosis. The tissue may be from a specific organ, such as liver, lung, colon, breast, prostate, etc. In one embodiment, the tissue is liver tissue.

[0102] The methods of the invention may comprise the further step of identifying a suitable enzyme whose activity or expression is downregulated in NASH compared to non-NASH tissues or whose activity or expression is upregulated in NASH compared to non-NASH tissues. The methods of the invention may comprise the further step of identifying substrates of the enzyme and optionally metabolites produced as a result of the action of the enzyme.

[0103] Therefore, the method described herein measures the activity of the enzyme by measuring the metabolism of the substrate in the biological sample, thereby indirectly measuring the activity of the enzyme in a non-invasive or minimally invasive manner, and the enzyme is directly related to the NASH disease state. Due to the correlation between the enzyme and its ability to decompose the substrate and the NASH disease state, the patient's disease state can be diagnosed or prognosed. On this basis, a suitable treatment method can be selected. In particular, the treatment can be selected from one or more of gastric bypass surgery and / or drug-based treatment, and the drug-based treatment includes the administration of at least one drug, and the at least one drug is selected from statins, incretin analogs, metformin, rimonabant, thiazolidinediones and orlistat.

[0104] In one embodiment, the method of the present invention includes a step of administering appropriate treatment to treat NASH disease after diagnosing the subject as having NASH or being at risk of developing NASH. Therefore, the present invention also provides a method for treating NASH in a subject, 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 GRAS compound, wherein the enzyme is an aldo-keto reductase (AKR) or an alcohol dehydrogenase, and comprising treating the subject.

[0105] The metabolism and conversion of a substrate by one or more enzymes results in the production of breakdown products, i.e., metabolites, i.e., metabolites. Soon after the substrate is provided to a subject, the substrate is excreted at high levels into a biological matrix, such as respiratory gas, urine, blood, and is removed from the biological matrix due to the biotransformation of the substrate by one or more enzymes (washout of reactants). For example, a kinetic curve for the removal of a substrate from respiratory gas can be used as a readout of the activity of the enzyme responsible for the biotransformation of the substrate.

[0106] In addition, the metabolism of a specific substrate by one or more enzymes results in the production of enzyme-specific metabolites. In this case, over time, the metabolites are excreted into the biological matrix, initially at lower levels, and over time, due to the biotransformation of the substrate by the enzyme. The measurement of such metabolites can be used as a probe to assess the metabolic phenotype of the one or more enzymes (enzyme or enzymes) responsible for the production of the product.

[0107] As further explained herein and in the Examples, the washout profiles of certain metabolites differ between NASH and non-NASH tissues, depending on whether the enzyme that metabolizes the substrate is overexpressed or downregulated in the NASH tissue.

[0108] For example, healthy hepatocytes showed higher production of 2-butanone (from the breakdown of butanol) at 6 hours, and as expected, the peak area of ​​healthy hepatocytes was larger than that of NASH, considering that in NASH, the alcohol dehydrogenase pathway involved in the conversion of 2-butanol to 2-butanone is downregulated (see Examples).

[0109] Regarding the production of nonanol from the breakdown of nonanal, NASH hepatocytes showed higher production of nonanol than healthy hepatocytes (peak area larger in NASH than in healthy). This result is consistent with the overexpression of AKR1B10 observed in NASH livers, the enzyme that converts nonanal to nonanol (see Examples).

[0110] In an embodiment, the enzyme used in the methods of the invention is an aldo-keto reductase (AKR). AKRs are enzymes that catalyze redox transformations involved in biosynthesis, intermediary metabolism, and detoxification.

[0111] AKRs catalyze redox reactions on a variety of substrates, including glucocorticoids, carbonyl metabolites, glutathione conjugates, and phospholipid aldehydes, among others (Barski, Tipparaju and Bhatnagar 2008. Aldo-Keto Reductase Superfamily and Its Role in Drug Metabolism and Detoxification. Drug Metabolism Reviews 40 (4)). Given the diversity of biological substrates, it appears that AKRs may have a common function, namely, detoxifying aldehydes and ketones generated 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): 595-97). Using pyridine nucleotides as cofactors, most AKRs catalyze the reduction of aldehydes and ketones, and are relatively inefficient alcohol dehydrogenases (Barski, Tipparaju and Bhatnagar 2008).

[0112] Most of the energy required for carbonyl reduction by AKRs is obtained from nucleotide cofactor binding rather than substrate binding, resulting in efficient reduction of substrates even when loosely bound to the active site. This explains why some AKR families, such as AKR1 B family, can act on a wide range of substrates (Grimshaw 1992, Aldose Reductase: Model for a New Paradigm of Enzymic Perfection in Detoxification Catalysts. Biochemistry 31(42): 10139-45) and demonstrates the key role of AKRs as detoxification enzymes. The carbonyl group present in aldehydes is very reactive and can easily attack nucleophilic centers such as protein amino acids and membrane phospholipids.

[0113] Reduction of the aldehyde carbonyl group to an alcohol by AKR reduces the overall chemical reactivity of the molecule and is one of the mechanisms by which reactive aldehydes are detoxified from cells (Barski, Tipparaju, and Bhatnagar 2008). Lipid peroxidation can produce a variety of different toxic aldehydes, as ROS can oxidize any bisallylic 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. OxidativeMedicine and Cellular Longevity; Yin, Xu, and Porter 2011. Free Radical LipidPeroxidation: Mechanisms and Analysis. Chemical Reviews 111(10): 5944-72). In addition, aldehydes are considered to be the main byproducts of lipid peroxidation (Barski, Tipparaju, and Bhatnagar 2008). Broad substrate specificity, as well as the ability to readily reduce reactive aldehydes, make the AKR family an ideal antioxidant mechanism for protecting against lipid peroxidation and ferroptosis. In support of this hypothesis, AKRs have been shown to reduce lipid peroxidation products 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)).

[0114] Members of the AKR1 B family have been shown to act on several highly volatile compounds. In fact, AKR1B1, AKR1 B10, and AKR1 B15 have been shown to display substrate specificity for the volatile aldehydes benzaldehyde and cinnamaldehyde, the alkanals hexanal, the alkenals 4-hydroxynonenal, hexenal, and farnesaldehyde, the ketone 3-nonen-2-one, the dicarbonyl 2,3-butanedione, and 2,3-hexanedione (Gimenez-Dejoz et al., 2015).

[0115] Aldehyde-keto reductase family 1 member B10 (AKR1B10) is associated with HCC and is secreted into the blood by hepatocytes via a lysosome-mediated non-classical pathway. Secretion of AKR1B10 protein is associated with advanced NASH (Kanno, M. et al. 2019. Serumaldo-keto reductase family 1 member B10 predicts advanced liver fibrosis and fatal complications of nonalcoholic steatohepatitis. J Gastroenterol 54, 549-557).

[0116] In an embodiment, the enzyme may be an AKR family 1 member, in particular it may be AKR family 1 member B10 (AKR1B10).

[0117] In another embodiment, the enzyme used in the methods of the invention is an alcohol dehydrogenase.Alcohol metabolism is a well-characterized biological process dominated by the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) families.

[0118] Alcohol dehydrogenase catalyzes the oxidation of primary and secondary alcohols to the corresponding aldehydes or ketones. Changes in alcohol metabolism in response to human NASH progression have been studied, as have the activity and expression of alcohol dehydrogenases (Li, H., Toth, E. & Cherrington, NJ Alcohol Metabolism in the Progression of Human NonalcoholicSteatohepatitis. Toxicol Sci 164, 428-438, (2018)).

[0119] In an embodiment, the enzyme is αα-alcohol dehydrogenase (ααADH). In an embodiment, the enzyme is ALDH4A1, ADH1A, ADH1B, ADH4 and ALDH2.

[0120] The exogenous substrate used in the method of the present invention is specific for the enzyme, so that the substrate is selectively metabolized by the enzyme. Therefore, the exogenous substrate can be any substrate suitable for detecting enzyme activity. In an embodiment, the substrate and / or its metabolite are VOCs secreted into the biological matrix, preferably VOCs secreted into the biological matrix at a high ratio. In general, VOC is defined as an organic compound, the composition of which makes it possible to evaporate under normal indoor temperature and pressure atmospheric conditions. Since the lower the boiling point temperature of the compound, the higher its volatility is usually, the volatility of the organic compound is sometimes defined and classified by its boiling point. Volatile compounds are compounds secreted into gaseous liquids by the human body, and the gaseous liquids include, for example, respiratory gases, skin emanations, etc. Optionally, the substrate and / or metabolite are such VOCs, and the VOCs can be measured in a biological matrix without the use of any labeling such as isotope labels. Preferably, the exogenous substrate can be selected from aldehydes and / or alcohols.

[0121] 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 respiratory gas is measured.

[0122] Therefore, the method uses exogenous volatile organic compounds (EVOCs) as tracers of specific in vivo liver-specific metabolic activity. EVOCs can be volatile compounds that are administered to a subject by various routes, undergo metabolism and distribution in the body, and are excreted through respiration. In addition, the metabolism of EVOCs by liver-specific enzymes can lead to the production of other volatile compounds that can also be detected in the breath.

[0123] 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 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 breath is measured.

[0124] If the substrate is a VOC, it may or may not be labeled.

[0125] The VOCs measured according to the method may not be naturally occurring / produced by the subject and excreted into the biological matrix. This ensures that any readings are not contaminated by endogenous VOCs that are naturally occurring and can be found in biological matrices.

[0126] In one embodiment, the substrate is a naturally occurring compound (but not endogenously produced), such as a food compound. This has the advantage that it can be provided to a subject without the occurrence of side effects. In one embodiment, the substrate does not have any therapeutic benefit. In one embodiment, the substrate is not a non-naturally occurring compound.

[0127] In one embodiment, the substrate is a GRAS compound, such as a GRAS compound that is a VOC. "GRAS" is an acronym for the phrase "generally recognized as safe". According to 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 pre-market review and approval by the FDA unless the substance is recognized among qualified experts as being sufficiently proven to be safe under the conditions of its intended use, or unless the use of the substance does not fall within the definition of a food additive. For example, the GRAS compound can be a naturally occurring compound. For example, the GRAS compound can be selected from a food or a food additive. In one embodiment, the GRAS compound is a vitamin, a phenolic flavoring, 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 substance primarily used for flavoring, coloring, or preserving food. In one embodiment, the GRAS compound is an aliphatic or aromatic terpene hydrocarbon or terpenoid. In one embodiment, the GRAS compound is an EU-approved food flavoring.

[0128] In one embodiment, the substrate is not a VOC and its metabolite is not a VOC. In this embodiment, the substrate is a labeled reactant, and the labeled reactant and / or labeled metabolite can be measured in breath. The label can be an isotopic label, such as 12C, 13C, 14C, 2H, 14N or 18O.

[0129] In an embodiment, the substrate and / or metabolite is a VOC and the substrate is not labeled. Therefore, no labeling is required because the substrate and / or metabolite can be measured in a biological matrix without using any labeling.

[0130] In an 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. If nonanal is used in the present method, 1-nonanal may also be used. 1-nonanal can be metabolized to 1-nonanol, and thus 1-nonanol may be a metabolite detected in the present method.

[0131] 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.

[0132] In one embodiment, the enzyme is an AKR enzyme, such as AKR1B10, the substrate is hexanal, and the metabolite is hexanol.

[0133] In one embodiment, the enzyme is an AKR enzyme, such as AKR1B10, the substrate is benzaldehyde, and the metabolite is benzyl alcohol.

[0134] In one embodiment, the enzyme is an AKR enzyme, such as AKR1B10, the substrate is citral, and the metabolite is nerol.

[0135] In 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 for steroid hormone synthesis and drug metabolism.

[0136] In an embodiment, the enzyme is CYP1A1, CYP1A2, CYP1 B1, 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.

[0137] 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.

[0138] 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. Figure 7The conversion of benzoic acid to hippuric acid is demonstrated in . In one embodiment, the enzyme is glycine N-acyltransferase. In one embodiment, the substrate is benzoic acid and the metabolite is hippuric acid.

[0139] In one embodiment, the enzyme is a CYP enzyme, the substrate is 2-pentanone, and the metabolite is 2,3-pentanediol.

[0140] In one embodiment, a substrate can produce a first metabolite and / or a first metabolite can be used as a substrate to produce a second metabolite. In some embodiments, the concentration of the substrate is measured. In some embodiments, the concentration of the first metabolite is measured. In some embodiments, the concentration of the second metabolite is measured. In some embodiments, the concentration of the substrate and the first metabolite is measured. In some embodiments, the concentration of the first metabolite and the second metabolite is measured. In some embodiments, the concentration of the substrate and the second metabolite is measured. In some embodiments, the concentration of the substrate, the first metabolite, and the second metabolite is measured.

[0141] 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 concentration of the metabolites 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 is measured. In one embodiment of the above method, the concentration of the substrate and the metabolite is measured.

[0142] In the above methods, the concentration of one or more substrates and / or one or more metabolites is measured. Thus, in one embodiment, the concentration of multiple metabolites is measured.

[0143] Thus, the methods provided herein are capable of detecting a variety of compounds in exhaled breath. This allows the detection of the presence of more than one disease. In addition, a variety of compounds specific to a certain type of disease can be measured in breath, thereby achieving a more accurate diagnosis due to the multiple parameters evaluated. Therefore, in one embodiment, the present invention relates to a method for detecting a disease, the method comprising evaluating the activity of one or more disease-specific enzymes by measuring the concentration of two or more exogenous substrates of the enzyme and / or measuring the concentration of two or more metabolites of the substrate(s) in the exhaled breath of a subject.

[0144] In one embodiment, the method disclosed herein for the detection of liver disease comprises evaluating the activity of more than one enzyme by measuring the concentration of two or more exogenous substrates of the enzyme and / or measuring the concentration of two or more metabolites of the substrate(s) in the exhaled breath of the subject. Thus, the method described herein can be a multiplex method, enabling the evaluation of multiple enzyme activities in the same breath sample(s) at the same time.

[0145] Therefore, on the one hand, the present invention relates to a method for detecting or prognosing NAFLD that progresses to the NASH stage in a subject, the method comprising measuring the concentration of an exogenous substrate of a 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 relates to a method for detecting or prognosing NAFLD that progresses to the NASH stage in a subject, the method comprising measuring the concentration of an exogenous substrate of AKR1B10 and / or measuring the concentration of a metabolite of the substrate in a biological sample obtained from the subject, wherein the substrate is nonanal. Further nonanal can be metabolized to nonanol, so in an embodiment, the metabolite is nonanol.

[0146] In an embodiment, the enzyme is an alcohol dehydrogenase, such as ααADH. In an embodiment, the substrate is selected from aldehydes and / or alcohols. In an embodiment, the enzyme is an alcohol dehydrogenase, the substrate is butanol and the metabolite is butanone.

[0147] In one embodiment, the enzyme is an alcohol dehydrogenase, and the substrate is 2-pentanone and the metabolite is 2-pentanol, 3-hydroxy-2-pentanone and / or 2,3-pentanediol.

[0148] In one embodiment, the enzyme is an alcohol dehydrogenase, and the substrate is benzyl alcohol and the metabolite is benzaldehyde and / or benzoic acid.

[0149] In one embodiment, the enzyme is aldehyde dehydrogenase, the substrate is benzaldehyde, and the metabolite is benzoic acid.

[0150] The present invention also relates to a method for detecting or prognosing NASH 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 GRAS compound, wherein the substrate is 2-pentanone and the metabolite is 2-pentanol, 3-hydroxy-2-pentanone and / or 2,3-pentanediol.

[0151] Therefore, in one aspect, the present invention relates to a method for detecting or prognosing NAFLD that progresses to the NASH stage in a subject, the method comprising measuring the concentration of an exogenous substrate of a 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 butanol. Preferably, the present invention relates to a method for detecting or prognosing NAFLD that progresses to the NASH stage in a subject, the method comprising measuring the concentration of an exogenous substrate of ADH and / or measuring the concentration of a metabolite of the substrate in a biological sample obtained from the subject, wherein the substrate is butanol. Further, butanol can be metabolized to butanone, so in an embodiment, the metabolite is butanone.

[0152] If butanol is used in the method of the present invention, 2-butanol may also be used. 2-Butanol can be metabolized to 2-butanone, and thus 2-butanone may be a metabolite detected in the method of the present invention.

[0153] In the above methods, the concentration of one or more substrates and / or one or more metabolites is measured. Thus, in one embodiment, the concentration of multiple metabolites may be measured.

[0154] By using the hepatocyte model described in the examples, which is a model of early liver disease, the inventors have developed a method that can distinguish NASH from other liver disease stages and thus enable early diagnosis and intervention. This distinction relies on changes in enzyme activity induced by NASH, which leads to changes in the metabolic rate of specific compounds. Quantification of these compounds allows the identification of subjects with NASH.

[0155] In another aspect, the present invention relates to a method of distinguishing 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 GRAS compound.

[0156] In another aspect, the present invention relates to a method for detecting or prognosing early-stage NASH 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 GRAS compound.

[0157] In another aspect, the invention relates to a method of determining the stage of NASH 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 GRAS compound.

[0158] As described above, in these methods, the activity or expression of the enzyme is upregulated or downregulated in NASH. For example, the enzyme may be AKR or ADH, as further explained above. For example, the enzyme may be AKR, and the substrate and metabolite may be selected from those listed above. For example, respectively, the substrate may be selected from nonanal, trans-2-hexenal, hexanal, benzaldehyde, citral, and the corresponding metabolite is selected from 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, respectively, the substrate may be selected from butanol or 2-pentanone, and the corresponding metabolite is selected from butanone, 2-pentanol, 3-hydroxy-2-pentanone or 2,3-pentanediol.

[0159] In another embodiment, the enzyme is a CYP enzyme. The cytochrome CYP450 (CYP450) enzyme family is responsible for the metabolism of most drugs and lipophilic xenobiotics and is therefore of great importance in clinical pharmacology. Although several different families of CYP450 enzymes exist in the human body, enzymes belonging to families 1-, 2-, and 3- are involved in the metabolism of the vast majority of administered therapeutic drugs.

[0160] These enzymes convert prodrugs into the corresponding biologically active compounds and convert active drugs into inactive metabolites, which are subsequently excreted from our bodies. Differences in the enzymatic activity of these enzymes lead to differences in the biotransformation of xenobiotics, ultimately resulting in toxicity of the compounds or ineffectiveness of the drugs. Many factors contribute to the diversity of CYP450 activity and, therefore, to the diversity of their metabolic efficiency.

[0161] In one embodiment, the CYP450 enzyme is selected from family 1, 2 or 3. For example, the CYP450 enzyme is selected from CYP1A1, CYP1A2, CYP1 B1, 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-glucuronosyltransferase or aldehyde dehydrogenase.

[0162] 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.

[0163] In one embodiment, the enzyme is glycine N-acyltransferase. In one embodiment, the enzyme is glycine N-acyltransferase, the substrate is benzoate, and the metabolite is hippurate.

[0164] The method needs to measure the concentration of exogenous substrates and / or their metabolites in the biological sample from the subject, i.e. the test subject. The term biological sample can be used interchangeably with the term biological matrix. In embodiments, the biological sample or matrix is ​​selected from respiratory gas, 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 embodiments, the biological sample is a body fluid sample. In the art, methods for obtaining body fluid samples are well known. In embodiments, the body fluid sample can be a sample of blood, urine or exhaled gas. The blood sample can include one or more of plasma, red blood cells, white blood cells, platelets. The blood sample can include any combination of plasma, red blood cells, white blood cells, platelets.

[0165] In one embodiment, the sample is exhaled breath.

[0166] When the body fluid sample is a sample of exhaled gas, the respiratory gas sample may include air exhaled from one or more different parts of the subject's body (such as the nostrils, pharynx, trachea, bronchioles, alveoli, etc.). For the collection and measurement of respiratory samples, the devices and methods described in WO2017 / 187120 or WO2017 / 187141 (both publications are incorporated herein by reference) can be used.

[0167] In embodiments where the biological sample is a sample of exhaled breath, this can be obtained by collecting air exhaled by 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 blocks gas from infiltrating into and out of the bag and / or is chemically inert, thereby ensuring sample integrity. Exhaled gas can also be collected using a breath collection device. Preferably, the collection of the sample of exhaled breath is performed in a minimally invasive or non-invasive manner.

[0168] Determination of the amount of one or more VOCS in a subject's exhaled breath sample may be performed using at least one technique including, but not limited to, gas chromatography (GC), gas chromatography tandem 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), an electronic nose device, a quartz crystal microbalance, or a chemically sensitive sensor.

[0169] The amount of one or more VOCS in a subject's exhaled breath sample can be determined using thermal desorption-gas chromatography-time of flight-mass spectrometry (GC-fof-MS). In a specific embodiment, the subject's breath is collected in an inert bag, and the contents of the bag are then transported to a desorption tube under standardized conditions, and the VOCs are analyzed by the contents of the thermal desorption tube and then separated by capillary gas chromatography. The volatile organic peaks are then detected by mass spectrometry and identified using, for example, a library of the National Institute of Standards and Technology. Thermal desorption can be performed at a temperature of, for example, about 200-350° C. at the GC inlet. In all chromatography methods, separation occurs when a sample mixture is introduced (injected) into a mobile phase. Gas chromatography (GC) typically uses an inert gas, such as helium, as a mobile phase. GC / MS allows separation, identification, and / or quantification of individual components from a biological sample. 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), thermal spray, 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 in a subject's exhaled breath sample.

[0170] In one embodiment, the method comprises collecting different selected exhaled breath samples or portions thereof on a single breath sample capture device, the method comprising the steps of:

[0171] (a) collecting a first exhaled breath sample by contacting the sample with a capture device comprising an adsorbent material;

[0172] (b) collecting a second exhaled breath sample by contacting the second sample with the capture device, wherein the first and second exhaled breath samples are captured on the capture device in a spatially separated manner.

[0173] Therefore, the present invention also relates to a method comprising:

[0174] (a) administering a substrate as described herein;

[0175] (b) collecting a first exhaled breath sample by contacting the sample with a capture device comprising an adsorbent material;

[0176] (c) collecting a second exhaled breath sample by contacting the second sample with the capture device, wherein the first and second exhaled breath samples are captured on the capture device in a spatially separated manner.

[0177] 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-oxypropylene 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. A Carbograph® adsorbent is Carbograph 5TD. In one embodiment, the capture device comprises both Tenax GR and Carbograph 5TD. The capture device is conveniently an adsorbent tube. These are hollow metal cylinders, typically of standard size (3½ inches long, ¼ inch inside diameter), packed with a suitable adsorbent material.

[0178] A non-NASH subject refers to a subject that does not have NASH but may or may not have NAFL. A non-NASH subject also includes a healthy subject. As used herein, a "healthy subject" is defined as a subject that does not have the target NASH disease, i.e., a human being.

[0179] As used herein, "reference value" refers to a value determined by performing a test method on a plurality of reference subjects. The reference subject may be a healthy subject or a subject diagnosed with a disease.

[0180] "Likelihood of a disease state" refers to the probability that the disease state is present in a sample from a subject is about 50% or more, such as 60%, 70%, 80% or 90%.

[0181] As used herein, a decrease or increase can be 5%, 10%, 20%, 30%, 40%, 50% or more or at least or about 2 times, at least or about 2.5 times, at least or about 3 times, at least or about 3.5 times, at least or about 4 times, at least or about 5 times, at least or about 6 times, at least or about 7 times, at least or about 8 times, at least or about 9 times, or at least or about 10 times.

[0182] The methods of the present invention include determining the concentration of a substrate and / or metabolite in a breath sample of a test subject and then comparing the 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 a non-NASH subject who does not have or is destined to develop NASH.

[0183] The sample can be obtained at any time point. The sample can be obtained at any time point before or after the administration of the substrate (substrate(s)), such as 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 longer before or after treatment or therapy begins. The time point can also be earlier or later.

[0184] A change (increase or decrease) in the level of a substrate and / or metabolite relative to a baseline / reference value or range indicates 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 a metabolite in exhaled breath compared to a baseline value in a healthy individual indicates a risk of NASH. The AKR enzyme is overexpressed in NASH disease, and thus conversion of the substrate to the metabolite is increased. Conversely, a decreased level of the substrate in exhaled breath indicates a risk of NASH.

[0185] For example, in embodiments where the enzyme is ADH, healthy subjects have higher 2-butanone production than NASH patients because the alcohol dehydrogenase pathway involved in the conversion of 2-butanol to 2-butanone is downregulated in NASH.

[0186] The algorithm used to calculate the risk assessment score in the methods disclosed herein can group the concentration values ​​of substrates and / or metabolites, and the risk score can be derived from any algorithm known in the art. The algorithm is a set of rules that describe the risk assessment of NASH. The rule set can be defined only algebraically, but can also include alternative or multiple decision points that require specific field knowledge, expert interpretation, or other clinical indicators. Many algorithms that can provide different risk assessments can be developed using concentration curves of appropriate substrates and / or metabolites. For example, an individual's risk score can be generated using a Cox proportional hazard model. The prognostic classification of an individual can also be determined by using a statistical model or a machine learning algorithm that calculates the probability of recurrence based on the individual's substrate and / or metabolite concentrations.

[0187] Based on the determination of risk, individuals can be divided into risk groups (e.g., tertiles or quartiles) based on the numerical value of the selected risk score, where all individuals with numerical values ​​within a given range can be classified as belonging to a particular risk group. Thus, the selected numerical value will define the risk group of patients with greater or lesser risk, respectively. The risk groups can be further classified by different ranges of mortality, such as 6-month, 1-year, 2-year, 3-year, 4-year, 5-year, 10-year, 25-year mortality. The risk groups can be further classified by different ranges of events related to NASH, including but not limited to the likelihood of progression to NASH with fibrosis or NASH with cirrhosis.

[0188] The concentration of substrates and / or metabolites can be measured using methods known in the art. As used herein, concentration refers to the amount or mass of substrates and / or metabolites in a biological sample, for example, expressed in grams per liter (g / l). In one embodiment, the concentration is measured over time, for example, by measuring the kinetics of clearance. For example, the concentration is measured by evaluating a kinetic curve for clearance of the substrate (e.g., from respiration), which is then used as a readout. Additionally or alternatively, the excretion of metabolites that can be derived from the substrate can be measured over time. For example, the clearance of the substrate from the biological sample and the excretion of the metabolites can be measured in the same biological sample at the same time or at different times.

[0189] In one embodiment, the concentration or amount of a substrate and / or its metabolite can be determined in a plurality of biological samples in an absolute or relative manner, e.g. in a first breath sample (collected at a first time period) and in a second and / or further breath sample (collected at a later, second or further time period), thereby allowing analysis of its kinetics or rate of change of concentration over time.

[0190] In some embodiments, wherein the biological sample is an exhaled breath sample, 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-oxypropylene monomers. Carbograph® material is graphitized carbon black. In one embodiment, the material is Tenax GR, which comprises a mixture of Tenax®TA and 30% graphite. A Carbograph® adsorbent is Carbograph 5TD. In one embodiment, the capture device comprises both Tenax GR and Carbograph 5TD. The capture device is conveniently an adsorbent tube. These are hollow metal cylinders, typically of standard size (3½ inches long, ¼ inch inner diameter), packed with a suitable adsorbent material.

[0191] In one embodiment, the methods of the invention further comprise establishing a test subject value for one or more substrate and / or metabolite concentrations. The test subject value is compared to one or more reference (control) values, wherein a difference between the test subject value and the reference value indicates the likelihood of NASH.

[0192] In one embodiment, the reference value is from a non-NASH subject, such as a healthy subject.

[0193] In another embodiment, the reference value is from a subject diagnosed as having NASH (with or without fibrosis), or a subject whose NASH has progressed to cirrhosis or HCC.

[0194] The reference level of the test compound (i.e., substrate or metabolite) can be determined by measuring 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. The reference level can also be determined by measuring the level of the test compound in samples from patients before treatment.

[0195] In one embodiment, the method of the present invention further comprises comparing the subject value to one or more reference values. In one embodiment, the reference value is from a non-NASH subject, such as a healthy subject. In another embodiment, the reference value is from a subject diagnosed with NASH. In another embodiment, the reference value is a value for a subject who has been diagnosed with non-alcoholic fatty liver disease (NAFL). In another embodiment, the reference value is a value for a subject who has progressed to cirrhosis or HCC.

[0196] In one embodiment, the reference value is a NAFL subject value corresponding to a value calculated from a NAFL subject.In one embodiment, the presence of one or more subject values ​​greater than their respective healthy subject value ranges indicates a high likelihood of a NASH disease state in the test subject.

[0197] In one embodiment, the reference value is a healthy subject value corresponding to a value calculated from a healthy subject. In one embodiment, the presence of a magnitude of one or more subject values ​​greater than their respective healthy subject value ranges indicates a high likelihood of a NASH disease state in the test subject.

[0198] In one embodiment, an increase in the concentration of the test compound, e.g., substrate or metabolite, e.g., 5%, 10%, 20%, 30%, 40%, 50% or more compared to a reference value indicates a diagnosis of NASH or a risk that the subject will progress to NASH.

[0199] In one embodiment, a decrease in the concentration of a test compound (e.g., substrate or metabolite) by, e.g., 5%, 10%, 20%, 30%, 40%, 50% or more compared to a reference value indicates a diagnosis of NASH or a risk that the subject will progress to NASH.

[0200] In one embodiment, when the appropriate reference indicates that the subject does not have NASH, a detectable difference (e.g., a statistically significant difference) between a value determined from a subject in need of characterization or diagnosis of NASH and an appropriate reference may indicate NASH in the subject. In one embodiment, when the appropriate reference indicates NASH, a lack of a detectable difference (e.g., a lack of a statistically significant difference) between a value determined from a subject in need of characterization or diagnosis of NASH and an appropriate reference may indicate NASH in the subject. In an embodiment, an increase in metabolite concentration compared to a reference sample indicates NASH.

[0201] Thus, in one aspect, the method comprises detecting the concentration of substrates and / or metabolites in exhaled breath from a subject, and diagnosing the subject as having a likelihood of, or an increased risk of, a NASH disease state if the levels of one or more substrates and / or metabolites differ from healthy reference subject values.

[0202] Therefore, any of the methods described herein may further comprise the following steps:

[0203] a) comparing the amount of one or more VOCs in the biological sample to a reference value, wherein the reference value represents a known diagnostic, prognostic and / or monitoring state of NASH;

[0204] b) finding a deviation or no deviation of the amount of said one or more VOCs from said reference value; and

[0205] c) attributing the above biased or unbiased findings to a specific diagnostic, prognostic and / or monitoring status of NASH in the subject.

[0206] The term "deviation in amount" refers to an increase or decrease in the amount of one or more VOCs in a biological sample from a subject compared to a reference value. By "increased amount" we mean that the amount of said one or more VOCs in the biological sample from a subject is statistically higher than the reference value. By "decreased amount" we mean that the amount of said one or more VOCs in the biological sample from a subject is statistically lower than the reference value. An amount may be considered statistically higher or lower if its value differs from a predetermined threshold. The threshold may be, for example, the median of the amounts of VOCs determined in biological samples from a population of healthy subjects.

[0207] The term "unbiased amount" refers to a similar or unchanged amount of one or more VOCs of the present invention in a biological sample from the exhaled breath of a subject compared to a reference value. By "similar or unchanged level" is meant that the amount of the one or more VOCs in the biological sample from the subject is not statistically significantly different from the reference value. Preferably, the reference value is obtained from a sample of exhaled breath of one or more subjects of the same species, sex and age group as that in which NASH is measured, prognosed or monitored in the subject. Alternatively, the reference value may be a previous value of the amount of one or more VOCs obtained from a sample of exhaled breath of a particular subject. Such a reference value may be used if the method is used to monitor NASH, for example over time, or for monitoring the subject's response to a particular treatment.

[0208] The method may also include determining a risk score for the subject based on the concentration of the metabolite and / or substrate in the sample, and providing a prognosis for the subject using the risk score, wherein the risk score is indicative of the prognosis.

[0209] The method may comprise determining the concentration of two or more exogenous substrates of a NASH-specific enzyme and / or determining the concentration of two or more metabolites of said substrates.

[0210] In the method of the present invention, the subject has been administered an exogenous substrate for a NASH-specific enzyme. In a particular embodiment, the method may include the step of administering an exogenous substrate for a NASH-specific enzyme to the subject. Administration of the substrate may be by any reasonable route, including but not limited to oral, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitreal, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, and 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 subject is administered a substrate, the concentration of the substrate and / or its metabolites may be determined in a biological sample obtained from the subject.

[0211] One aspect relates to a method of monitoring the progression of NASH in a subject, the method comprising measuring the concentration of an exogenous substrate of a 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 and / or its metabolite is a VOC, and wherein the substrate is a GRAS compound, wherein the enzyme is an aldo-keto reductase (AKR) or an alcohol dehydrogenase as described above.

[0212] One aspect relates to a method for determining the efficacy of a treatment, comprising assessing the activity of an enzyme in a subject diagnosed with NASH 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 subject has been treated for NASH, wherein the enzyme is an aldo-keto reductase (AKR) or an alcohol dehydrogenase as described above.

[0213] The method for determining the effect of a treatment may include the following steps: (a) obtaining a first sample from a patient before the start of treatment or therapy (or at a first time point after the start of treatment or therapy, or when treatment or therapy starts); (b) determining the level of a substrate and / or metabolite in the first sample; (c) obtaining a second sample from the patient after the start of treatment or therapy (or at a second time point after the start of treatment or therapy); (d) determining the level of a substrate and / or metabolite in the first sample, and (e) comparing the level of a substrate and / or metabolite in the first sample with the level of a substrate and / or metabolite in the second sample.

[0214] Based on the level of the substrate and / or metabolite determined, if the level is increased or decreased compared to the level of the substrate and / or metabolite obtained in the first sample, the therapy is considered to be effective. An effective treatment or therapy can be continued, or if the patient's condition improves and no longer requires treatment, the effective treatment or therapy can be interrupted. An ineffective treatment can be changed or modified, or replaced with another treatment.

[0215] Treatment may include surgery or at least one drug selected from the group consisting of statins, incretin mimetics, metformin, rimonabant, thiazolidinediones, and orlistat.

[0216] In order to determine the effect of treatment, multiple samples can be obtained at different time points. Therefore, the method can include analyzing a first biological sample obtained from the subject at a first time point, and then analyzing one or more additional biological samples or ratios thereof obtained from the subject at one or more additional time points.

[0217] Thus, the method may further comprise the step of administering a treatment.

[0218] In an embodiment, the treatment of NASH is gastric bypass surgery and / or a drug-based treatment comprising administration of at least one drug selected from statins, incretin analogs, metformin, rimonabant, thiazolidinediones, and orlistat. Treatment of NASH is known in the art, see Ganguli et al., Heptat Med. 2019; 11: 159 - 178.

[0219] In one embodiment of either method, the substrate is not limonene. In one embodiment of either method, the substrate is not a CYP enzyme.

[0220] In some embodiments, the technology described herein is associated with a programmable machine designed to perform a series of arithmetic or logical operations provided by the methods described herein. For example, some embodiments of the technology are associated with computer software and / or computer hardware (e.g., implemented in). On the one hand, the technology relates to a computer including a form of memory, an element for performing arithmetic and logical operations, and a processing element (e.g., a microprocessor) for executing a series of instructions (e.g., methods provided herein) to read, operate, and store data. Therefore, a specific embodiment adopts the processing of data stored in or transmitted by one or more computer systems or other processing systems. The embodiment also relates to a device for performing these operations. Such a device can be specially constructed for the desired purpose, or it can also be a general-purpose computer (or a 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 perform some or all of the analysis operations collaboratively (e.g., through a network or cloud computing) and / or in parallel.

[0221] 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; generating a standard curve; determining the specificity and / or sensitivity of an assay or marker; calculating a ROC curve; sequence analysis; all as described herein or known in the art.

[0222] In some embodiments, the microprocessor is part of a system for: determining the amount of one or more substrates and / or metabolites associated with NASH, such as concentration; generating a standard curve; determining the specificity and / or sensitivity of an assay or marker; calculating a ROC curve; sequence analysis; all as described herein or known in the art. The amount of one or more substrates and / or metabolites can be determined by abundance, measured per mole or millimole.

[0223] The amount of one or more substrates and / or metabolites can be determined by assays known to those of skill in the art and described herein, including measurements using optical signals or other measurements known to those of skill in the art.

[0224] In some embodiments, a microprocessor or computer uses an algorithm to measure the amount of one or more substrates and / or metabolites. The algorithm may include mathematical interactions between marker measurements or mathematical transformations of marker measurements. Mathematical interactions and / or mathematical transformations may be represented in a linear, nonlinear, discontinuous, or discrete manner.

[0225] In some embodiments, a software or hardware component receives the results of multiple assays and determines a single value result reported to a user that indicates NASH disease risk based on the results of the multiple assays. Related embodiments calculate a risk factor based on a mathematical combination (e.g., a weighted combination, a linear combination) of the results from multiple assays as described elsewhere herein.

[0226] Compositions and kits

[0227] The present invention relates to a kit for detecting, diagnosing, screening or prognosing NASH, distinguishing NASH from NAFL or determining the efficacy of NASH treatment, which comprises nonanal and / or butanol, and a device for capturing a biological sample from a patient.

[0228] A kit as described herein may include a composition for administration comprising a substrate, i.e. nonanal, butanol, trans-2-hexenal, hexanal, benzaldehyde, citral or 2-pentanone or limonene. This may be formulated for oral administration, e.g. as a tablet or capsule.

[0229] The kit may include instructions for assessing or monitoring NASH in a patient based on target substrate and / or metabolite levels. In some embodiments, the kit comprises reagents for measuring target substrate and / or metabolite levels.

[0230] The components of the test kit can be packaged in an aqueous medium, or packaged in a lyophilized form. The container device of the test kit generally includes at least one vial, test tube, flask, bottle, syringe or other container device, in which the components can be placed, and preferably, appropriately aliquoted samples. When there is more than one component in the test kit, the test kit will generally also include a second, third or other additional container, in which additional components can be placed separately (for example, sterile, pharmaceutically acceptable buffer and / or other diluents). However, a combination of various components can be included in a vial. The test kit of the present invention will generally also include a device for accommodating nucleic acid, and any other reagent container for commercial sale that is sealed. This container can include an injection or blow molding plastic container, in which the required vial is retained.

[0231] The components of the kit are used for administration as described above. It may also include a pharmaceutically acceptable carrier or vehicle. This may be a granule, so that the composition is, for example, in the form of a tablet or powder. The term "carrier" refers to a diluent, adjuvant or excipient that is administered with a substrate. These pharmaceutical carriers may be liquids, such as water and oil, including liquids of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. The carrier may be saline, gum arabic, gelatin, starch paste, talcum powder, keratin, colloidal silica, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants may be used. Saline solutions and aqueous glucose and glycerol solutions may also be used as liquid carriers, particularly injectable solutions. Suitable pharmaceutical carriers also include excipients, such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum powder, sodium chloride, dry skim milk, glycerol, propylene, ethylene glycol, water, ethanol, etc. If necessary, the composition may also contain a small amount of a wetting agent or emulsifier or a pH buffer. The composition may be in the form of a liquid, such as a solution, emulsion or suspension. The liquid may be used for injection, infusion (e.g., intravenous infusion) or subcutaneous delivery. As a solid composition for oral administration, the composition may be formulated into a powder, granules, compressed tablets, pills, capsules, chewing gum, wafers or similar forms. The composition may be in the form of one or more dosage units.

[0232] The substrate may be included in a composition such as a nutritional supplement. Different absorption rates of the substrate into the blood can cause significant changes in the time to maximum concentration during breathing. Therefore, in one embodiment, the substrate is provided in a formulation to ensure rapid delivery. In one embodiment, the substrate is formulated as a liquid. In another embodiment, the substrate is formulated as a rapid release / fast dissolving tablet or capsule. This ensures that absorption has a shorter time constant than washout.

[0233] In another embodiment, the subject fasts overnight, and fasting can be combined with providing the substrate as a liquid or a rapid release / dissolving tablet or rapid release / dissolving capsule or other oral administration form.

[0234] Typically, the amount of substrate administered as part of the method of the invention or the amount of substrate included in the composition contained in the kit is at least about 0.01% of the substrate by weight of the composition. When intended for oral administration, the amount may vary in the range of about 0.1% to about 80% of the composition weight. For administration by injection, the composition may include about typically about 0.1 mg / kg to about 250 mg / kg of the subject's body weight, preferably between about 0.1 mg / kg to about 20 mg / kg of the subject's body weight, and more preferably about 1 mg / kg to about 10 mg / kg of the subject's body weight.

[0235] use

[0236] The present invention also relates to the use of an exogenous substrate for an enzyme in any of the methods described herein. In an embodiment, the exogenous substrate is selected from nonanal, butanol, trans-2-hexenal, hexanal, benzaldehyde, citral and / or 2-pentanone.

[0237] 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 a method for detecting, staging, monitoring or prognosing NASH in a subject, the method comprising 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 detecting, screening, monitoring, diagnosing or prognosing NASH in a subject in an in vivo, in vitro or ex vivo method.

[0238] One aspect of the 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 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, 3-hydroxy-2-pentanone and / or 2,3-pentanediol as biomarkers for one or more of NASH without fibrosis, NASH with fibrosis or NASH with cirrhosis.

[0239] 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 a method of 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.

[0240] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Although the foregoing disclosure provides a general description of the subject matter contained within the scope of the present invention, including methods of making and using the present invention and the best mode thereof, the following examples are provided to further enable those skilled in the art to implement the present invention and provide a complete written description thereof. However, it will be understood by those skilled in the art that the specific details of the embodiments should not be construed as limitations of the present invention, and their scope should be understood from the claims appended hereto and their equivalents. In view of the present invention, different further aspects and embodiments of the present invention will be apparent to those skilled in the art.

[0241] All documents mentioned in this specification are incorporated herein by reference in their entirety.

[0242] As used herein, "and / or" should be considered as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" should be considered as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, equivalent to each of the items being listed individually herein. Unless the context dictates otherwise, the descriptions and definitions of the above features are not limited to any particular aspect or embodiment of the invention, and apply equally to all aspects and embodiments described. DETAILED DESCRIPTION

[0243] The present invention is further described in the following non-combining examples.

[0244] Example

[0245] Example 1 Identification of differentially expressed genes in NASH and healthy / NAFLD tissues.

[0246] Gene expression datasets were obtained from dedicated repositories. These datasets were generated from liver tissues of healthy subjects or subjects affected by NAFLD ranging from simple steatosis to NASH to fibrosis. Analysis of genes differentially expressed between subjects with NASH and subjects with simple steatosis or healthy subjects identified AKR1B10 as a gene upregulated in NASH at the transcriptome level. Additional exploration of the available literature showed that AKR1B10 is also increased in NASH at the protein level.

[0247] Example 2 Production of Biological Products in Cell-Based Assays

[0248] The identified substrates of AKR1B10 are nonanal, trans-2-hexenal, hexanal, benzaldehyde, citral. We have tested the conversion of these substrates to the respective bioproducts 1-nonanol, trans-2-hexenol, hexenol, benzyl alcohol and nerol in cancer cell lines overexpressing AKR1B10. Addition of substrates to the culture medium resulted in conversion to the bioproducts within a 6-hour time frame. Elimination of AKR1B10 or treatment of cells with AKR inhibitors resulted in reduced or absent production of the bioproducts.

[0249] A549 lung cancer cells were targeted by the CRISP / Cas system to eliminate the protein expression of AKR1B10. Specific sgRNA for the AKR1B10 gene was co-transfected into the cells with SpCas9 using ribonucleoproteins (RNPs). Clonal cells were generated by using a limiting dilution method. After expansion, clones were screened using PCR, and a total of 4 correctly edited clones were identified. The resulting protein extracts of WT and AKR1B10 KO cells were analyzed using the Jess system and probed with an AKR1B10-specific antibody and an anti-α-tubulin antibody as independent reporters. Western blot analysis of cell extracts obtained from wild-type (WT) or AKR1B10 CRISP / Cas knockout (KO) cells yielded 4 different clones. The lack of AKR1B10 antibody staining in KO cell lysates indicated complete and constitutive elimination of the enzyme. The results are shown in Figure 2. Figure 1 shown.

[0250] Use culture medium to transfer 5x10 4WT or three AKR1B10 KO clones at 10 cells / mL were seeded in 24-well plates and allowed to adhere and proliferate for 48 hours until cell confluence. Then, the cells were treated with 30µM nonanal or 10µM trans-2-hexenal. Aliquots of the culture medium were collected at 0.5, 1, and 3 hours after treatment. Headspace analysis was performed using Centri coupled to a GC-MS Orbi-trap system. Peak identification of nonanal, 1-nonanol, trans-2-hexenal, and trans-2-hexenol was performed by comparison with the peaks of reference standards. The results are shown in Figure 2 shown.

[0251] The addition of 30 µM nonanal to the culture medium and incubation for 3 hours shows the reduction of nonanal due to natural evaporation. The addition of culture medium supplemented with 30 µM nonanal to the WT or AKR1B10 KO cell lines increased the reduction of nonanal over the 3 hours, indicating that the cells are metabolizing nonanal ( Figure 2 A). Under all conditions, nonanal was undetectable if only methanol was added to the medium. Figure 2 In B, the same medium was used Figure 2 A, The medium was supplemented with 30 µM nonanal. This shows that 1-nonanol is not produced in the absence of cells. Addition of medium to WT or KO cells shows 3-fold higher levels of 1-nonanol production in WT cells compared to KO cells. If nonanal is not added to the medium under all conditions, no 1-nonanol is detectable.

[0252] The addition of 10 µM trans-2-hexenal to the medium and incubation for 3 hours shows the reduction of trans-2-hexenal due to natural evaporation. The addition of medium supplemented with 10 µM trans-2-hexenal to the WT or AKR1B10 KO cell lines increased the reduction of trans-2-hexenal over 3 hours, indicating that the cells are metabolizing trans-2-hexenal. Under all conditions, trans-2-hexenal was undetectable if only methanol was added to the medium. Figure 2 D. Figure 2 The same medium used in C, supplemented with 10 µM trans-2-hexenal, shows that trans-2-hexenol is not produced in the absence of cells. Addition of medium to WT or KO cells shows that trans-2-hexenal is produced at a 10-fold higher level in WT cells compared to KO cells. If trans-2-hexenal is not added to the medium under all conditions, no trans-2-hexenol is detected.

[0253] The results showed that nonanal and trans-2-hexenal were substrates of AKR1B10, which were converted into 1-nonanol and trans-2-hexenol, respectively.

[0254] Example 3 Nonanal and 2-butanol washing experiment

[0255] The nonanal and 2-butanol washouts of the breath samples collected before and after the intake of 500 mg nonanal and 100 mg 2-butanol were analyzed for three volunteers in the study. Three breath collections were performed for each volunteer. For the three volunteers, the areas of nonanal and 2-butanol, as well as their respective bioproducts nonanol and 2-butanol, showed peaks of intensity within 20 minutes in the collected breath samples.

[0256] Three volunteers participated in the trial three times. They swallowed an emulsion containing 500 mg nonanal and 100 mg 2-butanol, and their breath was collected at eight different time points before and after ingestion of the emulsion using a ReCIVA, a mask with a breath collection tube for the storage of exhaled VOCs.

[0257] Appearance in breath and conversion of the probes to their bioproducts were measured by gas chromatography-mass spectrometry (GC-MS).

[0258] Nonanal and 2-butanol were detected in the breath samples of three volunteers collected after ingestion of the emulsion. The level of nonanal resulted in an increase within 20 minutes. Nonanol showed the same trend as nonanal, with a peak within 20 minutes after nonanal ingestion. The level of 2-butanol resulted in an increase within 20 minutes. 2-Butanone showed the same trend as 2-butanol, with a peak within 20 minutes after 2-butanol ingestion.

[0259] The results of the washout were as follows Figure 3 shown.

[0260] Example 4 Detection of VOCs from healthy human liver cells

[0261] Purpose

[0262] The metabolism of 2-butanol and nonanal by healthy human hepatocytes was studied over a 24-hour period and compared to changes in the concentrations of these compounds in a control plate without any cells over the same period of time.

[0263] Target

[0264] (a) Understand the metabolism of 2-butanol and nonanal in primary hepatocytes;

[0265] (b) to evaluate the changes in the levels of these compounds in culture medium with hepatocytes and culture medium without hepatocytes within 24 hours from the peak formation and compare them;

[0266] (c) Evaluation of the metabolic degradation rate of specific VOCs by healthy hepatocytes.

[0267] method

[0268] Using organ-on-a-chip samples.

[0269] The Organ Chip is a three-dimensional hepatocyte culture system developed by a company called CN-Bio Co., Ltd. Aliquots of the VOC-spiked culture medium collected at different time points were analyzed using CENTRI-GC-MS, a system for headspace detection and quantification of VOCs. The experiment contained samples from healthy hepatocytes in culture medium exposed to 2-butanol, nonanal at one of the following concentrations: 50, 10, 2, and 0 ng / µL, and a control medium without hepatocytes containing the same concentrations of 2-butanol and nonanal.

[0270] The system suitability test (SST) and 2 standard checks (0, 2, 10, and 50 ng / µL) were run in duplicate at the beginning, middle, and end of the sequence to mitigate the risk of instrument bias.

[0271] Empty tubes were also run at the beginning, middle, and end of the sequence to mitigate the risk of sample carryover, and 0.5 µL of internal standard (1000 ng / µL) was added to each sample prior to analysis.

[0272] in conclusion

[0273] Samples from plates containing media with hepatocytes and media treated with 2-butanol and nonanal alone were analyzed. eVOCs of 50, 10, 2, and 0 ng / µL were used. Instrument performance was checked and showed good agreement throughout the sequence. 2-Butanol and nonanal showed a decrease in both media alone and media with hepatocytes. When exposed to media with hepatocytes, metabolic rates were observed in all compounds, with only the media with 1-nonanol (a bioproduct of nonanal) and 2-butanone (a bioproduct of 2-butanol) showing a peak and subsequent decrease corresponding to the metabolism of nonanal and 2-butanol. Figure 4 and Figure 5 shown.

[0274] Example 5 Detection of VOCs from healthy and diseased human hepatocytes

[0275] Organ Chips containing healthy human hepatocytes as well as human hepatocytes that were manipulated to produce healthy or NASH liver disease models were used in the following experiments. The Organ Chip containing healthy human hepatocytes was similar to the Organ Chip described above, but this experiment also included a NASH hepatocyte model. The Organ Chip was as described above, but this experiment also included a NASH hepatocyte model. The latter was obtained by treating healthy hepatocytes with Kupfer cells and hepatic stellate cells. These additional cells induced inflammation and fibrosis accumulation at levels similar to those observed in NASH livers. The model has been validated by gene expression and protein expression and shows features consistent with those observed in NASH liver biopsies from affected subjects.

[0276] As explained in more detail below, healthy and NASH hepatocytes showed differences in the metabolism of nonanal and 2-butanol. NASH hepatocytes overexpressing AKR1B10 showed higher production of 1-nonanol after treatment with nonanal compared to healthy hepatocytes. Conversely, given the reported downregulation of the alcohol dehydrogenase pathway in NASH, NASH hepatocytes showed reduced production of 2-butanone when treated with 2-butanol, as expected.

[0277] Primary human hepatocytes (PHHs), Kupffers cells (KCs) and hepatic stellate cells (HSCs) were seeded onto CN-Bio's PhysioMimixLC12 MPS plates. PHHs cells were seeded at 6x10 cells per well in CN-Bio's seeding medium. 5 HKC and HSC cells were 6x10 per well. 4 cells (required to induce NASH phenotype). Cells were maintained at a medium flow rate of 1 μl / s throughout the experiment. On days 4, 6, 8, 11, and 12 after seeding, aliquots of the medium were taken to measure LDH and albumin to assess cell health. On day 13 (24 hours after VOCs treatment), the final medium collection for analysis was performed. Scaffold constructs were fixed in 4% formaldehyde for Oil Red O staining to assess fat accumulation.

[0278] Lactate dehydrogenase (LDH) levels were measured using the CytoTox 96 cytotoxicity (LDH) assay kit. Albumin was measured using the AssayMax Albumin ELISA kit. IL-6 and TIMP-1 ELISAs (R&D DuoSet kits) were performed on media samples collected on days 11 and 13 (24 hours after administration) to assess inflammation and fibrosis levels. Figure 6 shown.

[0279] Measurement of lactate dehydrogenase levels (LDH) in the supernatant of healthy and NASH hepatocytes showed that levels decreased after several days in culture, indicating that cell stress due to the culture process was absent when the cells were treated with nonanal and 2-butanol (6A). Albumin levels in the supernatant of the hepatocytes showed that NASH hepatocytes produced less albumin than healthy hepatocytes, as expected given the disease phenotype also observed in humans ( Figure 6 B). Levels of inflammatory and fibrotic markers, interleukin 6 (IL-6) and TIMP metallopeptidase inhibitor 1 (TIMP-1), were measured in the supernatants before and after treatment with nonanal and 2-butanol ( Figure 6 C and 6D).

[0280] Microscopic images of cultured hepatocytes stained with Oil Red O to visualize lipids. Figure 7 As shown in the microscopy images in , NASH hepatocytes show the expected strong staining.

[0281] Figure 6 and Figure 7 The data presented demonstrate that hepatocytes were successfully cultured and developed both healthy and NASH phenotypes.

[0282] Primary hepatocytes were treated with 2-butanol and nonanal diluted in hepatocyte culture medium at a concentration of 50 ng / µL. Aliquots of the culture medium were collected at 0.5, 1, 2, 4, 6, and 24 hours. The remaining culture medium compound mixture that was not added to the cells was considered as the 0 time point reference. Headspace analysis was performed using a Centri coupled GC-MS Orbi-trap system, an automated system for collecting VOCs in the headspace, which is 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. p-values ​​< 0.05 were considered statistically significant.

[0283] Figure 8 A. 2-Butanol levels decreased over time in the absence of hepatocytes due to natural evaporation. However, the presence of NASH and healthy hepatocytes showed a more significant decrease at 24 h. Figure 8 B. 2-Butanone levels showed no increase in the absence of hepatocytes. Considering that in NASH, the alcohol dehydrogenase pathway involved in the conversion of 2-butanol to 2-butanone is downregulated 2 , as expected, healthy hepatocytes showed higher production of 2-butanone at 6 h, and the peak area was larger in healthy hepatocytes than in NASH. Figure 8 C.

[0284] Nonanal showed a decrease in the absence of hepatocytes due to natural evaporation. However, in the presence of hepatocytes, nonanal showed a more significant decrease. Figure 8 D. Nonanol was not produced in the absence of hepatocytes, however NASH hepatocytes showed higher production of nonanol than healthy hepatocytes (peak area was larger in NASH than healthy). This result is consistent with the overexpression of AKR1B10 observed in NASH livers, the enzyme that converts nonanal to nonanol 3 .

[0285] We demonstrate for the first time that NASH-induced metabolic changes are detectable using an exogenous volatile organic compound (EVOC) probe that is safe for human consumption and appears in breath gas along with its bioproducts after administration. These data support breath analysis using EVOC probes for induced volatileomics for NASH detection assays that can be used to diagnose / detect NASH, monitor progression, or prognose NASH.

[0286] References

[0287] 1. Endo, S. et al., Kinetic studies of AKR1B10, human aldose reductase-like protein: endogenous substrates and inhibition by steroids. Arch BiochemBiophys 487, 1-9, doi:10.1016 / j.abb.2009.05.009 (2009).

[0288] 2. Li, H., Toth, E. & Cherrington, NJ Alcohol Metabolism in theProgression of Human Nonalcoholic Steatohepatitis. Toxicol Sci 164, 428-438, doi:10.1093 / toxsci / kfy106 (2018).

[0289] 3. Kanno, M. et al., Serum aldo-keto reductase family 1 member B10predicts advanced liver fibrosis and fatal complications of nonalcoholicsteatohepatitis. J Gastroenterol 54, 549-557, doi:10.1007 / s00535-019-01551-3(2019).

[0290] Example 6 Detection of VOCs from NASH rat model

[0291] Several groups of 3 Wistar Han rats were fed a normal diet (ND) and a similar number of 3 rats were fed a choline-deficient high-fat diet (CDHFD), a treatment widely used to induce liver fibrosis in rodent models. 1 After 8 weeks, liver fibrosis was assessed and, as expected, CDHFD rats were found to have higher fibrosis compared to ND rats ( Fig. 9 A, B, and C). CDHFD rats showed less weight gain compared to ND rats ( Fig.10 ). Ten weeks after the start of the diet, benzyl alcohol (208 mg / Kg) or 2-butanol (440 mg / Kg) or 2-pentanone (160 mg / Kg) were orally administered to the rats, and blood samples (200 µl) were collected before and at the following time points after administration: 5, 15, 30 min, 1, 1.5, 2, 4, 8, 12, 24 h.

[0292] Blood samples were analyzed using gas chromatography-mass spectrometry (GC / -MS) or HPLC to measure the metabolite bioproducts benzoic acid, 3-hydroxy-2-butanone, 2-3-butanediol, and 2,3-pentanediol.

[0293] Benzoic acid is a bioproduct of the alcohol dehydrogenase pathway, which is further metabolized to hippuric acid. Chronic liver injury induces a decrease in hippuric acid metabolism 2 Consistent with these reports, we observed increased benzoic acid concentrations in the blood of CDHFD rats compared with ND rats after benzyl alcohol administration. In particular, benzoic acid was not present before administration. Levels in the blood increased from 5 to 240 min after administration, with higher concentrations found in CDHFD rats. After 480 min, blood concentrations reached undetectable levels in all rats ( Fig.11 ).

[0294] 2-Butanol is converted to 2-butanone by alcohol dehydrogenase. 2-Butanone is then converted to 3-hydroxy-2-butanone and 2,3-butanediol by CYP enzymes. 3,4 We administered 2-butanol and found that the concentrations of 3-hydroxy-2-butanone and 2,3-butanediol increased 240 minutes after administration. At 8 and 12 hours after administration (480, 720 minutes), these compounds showed higher levels in CDHFD compared with ND rats ( Fig.12 and 13 ).

[0295] 2-Pentanone is converted to 2,3-pentanediol by CYP 4 After administration of 2-pentanone, blood levels of 2,3-pentanediol were elevated after 1 h, and after 4 h in CDHFD rats compared with ND rats ( Fig.14 ).

[0296] These data suggest that NASH induces liver changes that alter the metabolism of the tested compounds. These changes can be used for diagnostic purposes.

[0297] Materials and methods

[0298] All bioanalytical experiments were performed by Wuxi AppTec (Nantong) Co., Ltd., No. 699, Huashi South Road, Qidong, Nantong, Jiangsu, China. Tel: 0513-83395562. A total of 18 Wistar Han rats were acclimated for 6 days. After this, 9 rats maintained a normal diet, whereas 9 rats were switched to a choline-deficient high-fat diet (CDHFD) (L-amino acid diet containing 45 kcal% fat and 0.1% methionine, no added choline and 1% cholesterol) (Research Diets, Cat. No. A16092003, https: / / researchdiets.com / en / formulas / a16092003). Rats had free access to food and water at all times unless otherwise specified. Body weight was measured weekly.

[0299] Eight weeks after the start of the diet, rats underwent a survival liver biopsy procedure. Rats were deeply anesthetized with 3% isoflurane, and the left lateral lobe was harvested from a longitudinal incision (3–4 cm) caudal to the xiphoid process, and a piece of liver was collected slightly to the left in the abdominal skin of the animal. At the end of the procedure, animals were subcutaneously administered tolidine (2 mg / kg) and penicillin (10 w U / rat), then once daily as needed for 2–3 days and monitored until full recovery. The collected liver tissue was sectioned, stained with hematoxylin / eosin, and the percentage of fibrotic tissue in the total area was quantified microscopically. Rats were allowed to recover from the biopsy for 2 weeks before further treatment.

[0300] After 10 weeks of dietary treatment, the rats were randomly divided into groups, each including 3 healthy rats and 3 NASH rats, and given different compounds.

[0301] Each group received an emulsion containing the reported compound in the indicated amounts in Table 1 .

[0302] Table 1: Compounds and doses administered.

[0303]

[0304] Each compound formulation was administered as an emulsion containing 15% Tween 80 diluted in water to deliver the target amount at 5 mL / kg body weight. The emulsion was administered by oral gavage after the rats were fasted for 16 h.

[0305] Blood samples were collected before compound administration (0 min) and at the following time points after administration: 5 min, 15 min, 30 min, 1 h, 1.5 h, 2 h, 4 h, 8 h, 12 h, and 24 h.

[0306] At each time point, approximately 200 μL of blood samples were collected into EDTA-K2 tubes and placed on wet ice until centrifugation. Plasma was generated from blood samples by centrifugation at approximately 4°C, 3200 g for 5 min.

[0307] 100 μL of plasma was collected and transferred into Eppendorf tubes. 95 μL of plasma was added with 400 μL of acetonitrile solution of internal standard, vortexed for 30 seconds, and centrifuged at 3220 g for 15 minutes. Unless otherwise specified, a total of 100 μL of supernatant was used for compound quantification.

[0308] The quantification of the target compounds was performed as shown in Table 2.

[0309] Table 2 - Quantification methods for target compounds.

[0310]

[0311] Details of the analytical method are as follows:

[0312] 3-Hydroxy-2-butanone (internal standard = acetone):

[0313] 2,3-Butanediol and 2,3-pentanediol (internal standard = 1-heptanol)

[0314] Table 3 – GC-MS analytical methods / parameters for the analysis of 3-hydroxy-2-butanone, 2,3-pentanediol, and 2,3-butanediol

[0315]

[0316] Benzoic acid (internal standard = hexanoic acid):

[0317] Inject directly into the instrument.

[0318] Table 4 - HPLC analysis method / parameters for benzoic acid analysis

[0319]

[0320] The response of each compound was normalized by the response of the internal standard, and absolute quantification was performed according to the calibration curve.

[0321] References

[0322] 1. Nevzorova YA, Boyer-Diaz Z, Cubero FJ, Gracia-Sancho J. Animalmodels for liver disease - A practical approach for translational research. JHepatol 2020;73(2):423-440. doi: 10.1016 / j.jhep.2020.04.011. PMID: 32330604

[0323] 2. Saltzman A, Caraway WT. Cinnamic acid as a test substance in theevaluation of liver function. J Clin Invest 1953;32(8):711-719. doi: 10.1172 / JCI102785. PMID: 13069619

[0324] 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

[0325] 4. Dietz FK, Rodriguez-Giaxola M, Traiger GJ, Stella VJ, HimmelsteinKJ. Pharmacokinetics of 2-butanol and its metabolites in the rat. JPharmacokinet Biopharm 1981;9(5):553-576. doi: 10.1007 / BF01061026. PMID:7334459

Claims

1. A method for detecting, staging, monitoring or prognosing non-alcoholic steatohepatitis (NASH) in a subject, the method comprising measuring the concentration of an exogenous substrate for 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, wherein 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.

2. The method of claim 1, wherein the method further comprises 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, wherein the method is used for detecting or prognosing NASH with fibrosis.

4. The method according to any one of the preceding claims, wherein the enzyme is aldo-keto reductase (AKR).

5. The method of claim 4, wherein the AKR is an AKR family 1 member. The method of claim 5 , wherein the AKR is AKR1B10.

7. A method according to any one of the preceding claims, wherein the substrate is selected from aldehydes and / or alcohols.

8. The method according to any one of the preceding claims, wherein the substrate is nonanal and / or the metabolite is nonanol.

9. The method of claim 8, wherein the substrate is 1-nonanal and / or the metabolite is 1-nonanol.

10. The method according to claims 1 to 7, wherein the substrate is trans-2-hexenal and / or the metabolite is trans-2-hexanol.

11. The method according to claims 1 to 7, wherein the substrate is hexanal and / or the metabolite is hexanol.

12. The method according to claims 1 to 7, wherein the substrate is benzaldehyde and / or the metabolite is benzyl alcohol.

13. The method according to claims 1 to 7, wherein the substrate is citral and / or the metabolite is nerol.

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

15. The method of claim 14, wherein the substrate is butanol and / or the metabolite is butanone.

16. The method of claim 15, wherein the substrate is 2-butanol and / or the metabolite is 2-butanone.

17. The method of claim 14, wherein the substrate is 2-pentanone.

18. The method of claim 14 or 17, wherein the substrate is 2-pentanone and / or the metabolite is 2-pentanol, 3-hydroxy-2-pentanone or 2,3-pentanediol.

19. The method of claim 14, wherein the substrate is benzyl alcohol and the metabolite is benzaldehyde and / or benzoic acid.

20. The method of claim 14, wherein the enzyme is aldehyde dehydrogenase.

21. The method of claim 20, wherein the substrate is benzaldehyde and the metabolite is benzoic acid.

22. The method of any one of claims 1 to 3, wherein the enzyme is a cytochrome P450 (CYP).

23. The method of claim 22, wherein the CYP is CYP1A1, CYP1A2, CYP1 B1, 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.

24. A method according to claim 22 or claim 23, wherein the substrate is 2-butanone and the metabolite is 3-hydroxy-2-butanone and / or 2,3-butanediol.

25. The method of claim 22 or claim 23, wherein the substrate is 2-pentanone and the metabolite is 2,3-pentanediol.

26. In one embodiment, the enzyme is glycine N-acyltransferase.

27. The method of claim 26, wherein the substrate is benzoic acid and the metabolite is hippuric acid.

28. A method according to any one of the preceding claims, wherein the substrate is labeled.

29. The method of claim 28, wherein the label is 12C, 13C, 14C, 2H, 14N or 18O.

30. The method according to any one of claims 1 to 27, wherein the substrate is unlabeled.

31. The method according to one of the preceding claims, wherein the biological sample is selected from respiratory gases, urine, blood, serum and / or tissue.

32. The method of one of the preceding claims, wherein the method comprises establishing the test subject value based on the concentration of the substrate or metabolite, or a combination thereof, in the test subject.

33. The method of claim 32, wherein the test subject value is compared to one or more reference values, wherein a difference between the test subject value and the reference value indicates a likelihood of NASH.

34. The method of claim 33, wherein the reference value is a value for a subject who has been diagnosed with NASH.

35. The method of claim 33, wherein the reference value is a value for a subject who has been diagnosed with non-alcoholic fatty liver disease (NAFLD).

36. The method of claim 33, wherein the reference value is a value for a subject with NASH that will progress to decompensated cirrhosis or HCC.

37. The method of claim 33, wherein the reference value is a value for a healthy subject.

38. The method according to one of the preceding claims, wherein the concentration of two or more exogenous substrates and / or the concentration of two or more metabolites of the enzyme are measured.

39. The method according to one of the preceding claims, wherein the subject has been administered an exogenous substrate for the enzyme.

40. The method according to one of the preceding claims, wherein the concentration of the metabolite is measured.

41. A method for determining the efficacy of a treatment, the method comprising assessing the activity of an enzyme in a subject diagnosed with NASH 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 subject has been treated for NASH, wherein the enzyme is aldo-keto reductase (AKR) or alcohol dehydrogenase or cytochrome P450 (CYP).

42. The method of claim 41, wherein the method comprises analyzing a first biological sample obtained from the subject at a first time point, followed by analyzing one or more additional biological samples obtained from the subject at one or more additional time points, or a ratio thereof.

43. The method of claim 41 or claim 42, wherein the treatment of NASH is gastric bypass surgery and / or a drug-based therapy comprising administration of at least one drug selected from statins, incretin analogs, metformin, rimonabant, thiazolidinediones, and orlistat.

44. A method for monitoring the progression of NASH 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, wherein the enzyme is an aldo-keto reductase (AKR) or an alcohol dehydrogenase or a cytochrome P450 (CYP).

45. A kit for the detection or prognosis of NASH, said kit comprising a substrate for an enzyme and / or a metabolite of said substrate and a device for capturing a biological sample from a patient.

46. ​​The kit of claim 45, wherein the substrate and / or metabolite is 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.

47. Use of exogenous substrates and / or metabolites of an enzyme in a method for detecting or prognosing NASH, wherein the activity or expression of the enzyme is upregulated or downregulated in 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.

48. 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 a method of detecting or prognosing NASH in a subject in vivo, 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.

49. 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.

50. A method of distinguishing 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.

51. The method of claim 50, wherein the activity or expression of the enzyme is upregulated or downregulated in NASH.

52. The method of claim 50 or claim 51, wherein the enzyme is an aldo-keto reductase (AKR) or an alcohol dehydrogenase or a cytochrome P450 (CYP).

53. A method for detecting or prognosing early-stage non-alcoholic steatohepatitis (NASH) 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.

54. The method of claim 53, wherein the enzyme is aldo-keto reductase (AKR) or alcohol dehydrogenase or cytochrome P450 (CYP).

55. A method of determining the stage of NASH 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.

56. The method of any one of claims 50 to 55, wherein the activity or expression of the enzyme is upregulated or downregulated in NASH.

57. The method of any one of claims 50 to 56, wherein the enzyme is an aldo-keto reductase (AKR) or an alcohol dehydrogenase or a cytochrome P450 (CYP).

58. A method for detecting or prognosing NASH in a subject, detecting or prognosing early NASH in a subject, determining the stage of NASH in a subject, or distinguishing NASH from other stages of NAFLD in a subject, 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, wherein the enzyme is not a CYP enzyme.

59. A method for detecting or prognosing NASH in a subject, detecting or prognosing early NASH in a subject, determining the stage of NASH in a subject, or distinguishing NASH from other stages of NAFLD in a subject, 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, wherein the substrate is not limonene.

60. A method for determining the efficacy of a treatment, the method comprising assessing the activity of an enzyme in a subject diagnosed with NASH 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 wherein the substrate is not limonene.

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