EZETIMIBE METABOLITE AS A NON-INVASIVE BIOMARKER FOR NON-ALCOHOLIC STEATOHEPATITIS (NASH)

Abstract

Method of utilizing ezetimibe-glucuronide (EZE-Gluc) as a diagnostic biomarker for a hepatic disorder is described herein. Non-alcoholic Fatty Liver Disease (NAFLD) is the most prevalent chronic liver disease, affecting 25% people worldwide and 64 million people in the United States. The initial stage of NAFLD is simple steatosis, which is characterized by microvesicular fat deposition. Approximately 10% of patients with steatosis progress to non-alcoholic steatohepatitis (NASH), which has significant clinical implications. At present, a liver biopsy is the only definitive way of diagnosing patients with NASH. Therefore, an alternate method of delineating NASH patients from those with steatosis is needed. EZE-Gluc can provide a non-invasive, specific, and selective way of identifying patients with NASH.

Claims

1.-33. (canceled)

34. A method for diagnosing a hepatic disorder in a human subject, die method comprising: a) administering ezetimibe (EZE) to the human subject; b) obtaining a sample from the human subject after a period of time from when the human subject was administered EZE; c) determining an amount of EZE, ezetimibe-glucuronide (EZE-Gluc), or both in the sample obtained from the human subject; d) comparing the amount of EZE, EZE-Gluc, or both to a control; and e) diagnosing the human subject with the hepatic disorder when the amount of EZE, EZE-Gluc, or both in the sample is elevated relative to the control.

35. The method of claim 34, wherein the sample is a blood sample, a urine sample, a serum, or a plasma sample.

36. The method of claim 34, wherein the human subject further has one or more of a metabolic syndrome, non-alcoholic fatty liver disease, obesity, dyslipidemia, insulin resistance, or diabetes.

37. The method of claim 34, wherein the hepatic disorder is non-alcoholic steatohepatitis, hepatic fibrosis, or hepatitis.

38. The method of claim 34, wherein the human subject further has, or previously had, one or more conditions selected from the group consisting of antitrypsin deficiency, Wilson's disease, fructosemia, galactosemia, Type III, IV, VI, IX, and X glycogen storage diseases, hemochromatosis, Gaucher's disease, Zellweger syndrome, tyrosinemia, bacterial infection, viral infection, parasitic infection, fungal infection, protozoan infection, helminth infection, spirochete infection, Budd-Chiari syndrome, alcoholism, drug addiction, heart failure, hepatic veno-occlusive disease, portal vein thrombosis, sarcoidosis, ulcerative colitis, or Crohn's disease.

39. The method of claim 34, wherein about 0.001 mg to about 10 mg of EZE per dose is administered to the human subject.

40. The method of claim 34, wherein the sample is obtained about 1 minute to about 360 minutes after EZE administration.

41. The method of claim 34, wherein the control is obtained from an aggregate population of asymptomatic individuals without the hepatic disorder, without risk of hepatic disorder, or without signs or symptoms of a hepatic disorder.

42. The method of claim 34, wherein the amount of EZE, EZE-Gluc, or both is elevated by at least 50% relative to the control.

43. A method of treating a hepatic disorder in a human subject in need of such treatment, the method comprising: a. administering ezetimibe (EZE) to the subject; b. obtaining, a non-biopsy, fluid sample after a period of time has passed from administering the EZE; c. measuring an amount of EZE, ezetimibe-glucuronide (EZE-Gluc), or both in the fluid sample; d. comparing the amount of EZE-EZE-Gluc, or both to a reference value, wherein a deviation of the amount of EZE, EZE-Gluc, or both from the reference value indicates a stage of the hepatic disease; and e. prescribing a therapy to the subject based on the stage of the hepatic disorder.

44. The method of claim 43, wherein the therapy prescribed to the subject comprises one or more of dietary changes, exercise regimens, lifestyle changes, surgical interventions, or use of medications.

45. The method of claim 43, wherein the human subject further has one or more of a metabolic syndrome, non-alcoholic fatty liver disease, obesity, dyslipidemia, insulin resistance, or diabetes.

46. The method of claim 43, wherein the hepatic disorder is non-alcoholic steatobepatitis, hepatic fibrosis, or hepatitis.

47. The method of claim 43, wherein the human subject further has, or previously had, one or more conditions selected from the group consisting of antitrypsin deficiency, Wilson's disease, fructosemia, galactosemia, Type III, IV, VI, IX, and X glycogen storage diseases, hemochromatosis, Gaucher's disease, Zellweger syndrome, tyrosinemia, bacterial infection, viral infection, parasitic infection, fungal infection, protozoan infection, helminth infection, spirochete infection, Budd-Chiari syndrome, alcoholism, drug addiction, heart failure, hepatic veno-occlusive disease, portal vein thrombosis, sarcoidosis, ulcerative colitis, or Crohn's disease.

48. The method of claim 43, wherein about 0.001 mg to about 10 mg of EZE per dose is administered to the human subject.

49. The method of claim 43, wherein the sample is obtained about 1 minute to about 360 minutes after EZE administration.

50. The method of claim 43, wherein the reference value is from an aggregate population of asymptomatic individuals without the hepatic disorder, without risk of hepatic disorder, or without signs or symptoms of a hepatic disorder.

51. The method of claim 43, wherein the amount of EZE, EZE-Gluc, or both deviates by at least 50% relative to the reference value.

52. A method for delineating non-alcoholic steatohepatitis (NASH) from simple steatosis in a human subject, the method comprising a) administering ezetimibe (EZE) to the human subject; b) determining an amount of ELF, ezetimibe-glucuronide (EZE-Gluc), or both in non-biopsy sample obtained from the human subject after a period of time from when the human subject was administered EZE; c) comparing the amount of ELF, EYE-Glue, or both to a control, wherein the control is obtained from a human subject with steatosis; and d) determining that the human subject has NASH when the amount of EZE, EZE-Gluc, or both is elevated relative to the control.

53. The method of claim 52 further comprising measuring an amount of one or a combination of CK-18, perilipin-1 (PLIN-1), and PLIN-2 droplet proteins, and comparing said amount to a protein reference value, age, weight, ALT, AST, triglyceride, diabetes, hepascore, hyaluronic acid, elastography, acoustic radiation, fibroscan, MRI, CT, or PET scan.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0031] Referring now to FIGS. 1-2, the present invention features methods for the diagnosis and monitoring of a hepatic disorder using the metabolic biomarker, ezetimibe (EZE) and glucuronidated-ezetimibe (EZE-Gluc).

[0032] Referring now to FIG. 1A, one embodiment of a drug elimination pathway of a healthy liver is shown, where a drug such as EZE is glucuronidated by UDP-glucuronosyltransferase (UGT) in enterocyte and hepatocytes.

[0033] Referring now to FIG. 1B, one embodiment of drug elimination pathway of a NASH liver is shown. A shift in the preferential elimination pathway away from bile towards the blood is seen when EZE is given to a NASH patient. Without wishing to limit the present invention, MRP transporters MRP2 and MRP3 may alter human transporter function in livers of NASH versus healthy patients.

[0034] According to some embodiments, the present invention provides a method for diagnosing a hepatic disorder. In one embodiment, the method comprises determining an amount of EZE and/or EZE-Gluc in a plasma sample and/or a urine sample obtained from a human subject after the human subject was administered EZE, and comparing the amount of EZE and/or EZE-Gluc in one or both of the plasma sample and the urine sample of a control. The control or reference value is obtained from healthy individuals without a hepatic disorder, without signs/symptoms of a hepatic disorder, or without a risk for a hepatic disorder. In some embodiments, the subject can be identified as having a hepatic disorder if the amount of EZE and/or EZE-Gluc in one or both of the plasma sample and the urine sample is elevated relative to the control (e.g., compare plasma EZE-Gluc levels in human subject to plasma EZE-Gluc levels in control). In some embodiments, if the subject is below the reference threshold, the subject may not have hepatic disorder. In some embodiments, if the subject approaches the reference value, then the subject is at risk of developing the hepatic disorder. In some embodiments, if the subject surpasses the reference value, then the patient has the hepatic disorder. In some embodiments, if the subject surpasses a higher reference value, then the subject can be classified as having a more severe form of the hepatic disorder. In some embodiments, the amount of elevated EZE and/or EZE-Gluc levels is at least 50%, at least 100%, at least 200%, at least 300%, or at least 400% of control. In other embodiments, the amount of elevated EZE and/or EZE-Gluc is at least 10 times, 100 times, 1000 times, 10,000 times 100,000 times that of the control.

[0035] In some embodiments, the human subject may be at risk of, or diagnosed with, a hepatic disorder such as hepatitis, NAFLD, or NASH. Non-limiting examples of hepatic disorder co-morbidities may include metabolic syndrome, obesity, dyslipidemia, insulin resistance, hepatic fibrosis, and diabetes.

[0036] In some embodiments, the human subject may suffer from, or had previously suffered from, one or more conditions including, but not limited to, antitrypsin deficiency, Wilson's disease, fructosemia, galactosemia, Type III, IV, VI, IX, and X glycogen storage diseases, hemochromatosis, Gaucher's disease, Zellweger syndrome, tyrosinemia, bacterial infection, viral infection, parasitic infection, Budd-Chiari syndrome, alcoholism, cirrhosis, drug addiction, heart failure, hepatic veno-occlusive disease, portal vein thrombosis, fungal infection, protozoan infection, helminth infection, spirochete infection, sarcoidosis, ulcerative colitis, and Crohn's disease. In other embodiments, the human subject is a bariatric surgery patient. In still other embodiments, the human subject suffers from, or previously suffered from a disorder such as alcoholism or drug addiction.

[0037] In some embodiments, the amount of EZE and/or EZE-Gluc is measured in a plasma sample. The plasma sample for measuring the amount of EZE and/or EZE-Gluc may be taken from the subject after a period of time from administration of EZE. For example, the plasma sample may be taken from the subject about 1-10 minutes after EZE administration. In other embodiments, the plasma sample may be taken about 10-30 minutes, about 30-60 minutes, about 60-90 minutes, or about 90-120 minutes. In still other embodiments, the plasma sample may be taken about 2-4 hours, about 3-5 hours, or about 4-6 hours after EZE administration.

[0038] In other embodiments, the amount of EZE and/or EZE-Gluc is measured in a urine sample. The urine sample for measuring the amount of EZE and/or EZE-Gluc may be taken from the subject after a period of time from administration of EZE. For instance, the urine sample may be taken from the subject about 60-360 minutes after EZE administration. In some embodiments, the urine sample may be taken about 1-2 hours, about 2-4 hours, about 3-5, or about 4-6 hours after EZE administration.

[0039] It is to be understood that the present invention is not limited to the aforementioned times. In preferred embodiments, the amount of time between administering EZE to the subject and taking the plasma and/or urine sample is sufficient to allow for EZE and/or EZE-Gluc to be detected and levels thereof measured by any appropriate system, e.g., LC, MS, LC/MS, immune assays.

[0040] In some embodiments, the subject may be administered about 0.001 to 10 mg of EZE per dose. In other embodiments, the subject may be administered about 0.001 to 0.01 mg, about 0.01 to 0.1 mg, about 1.0 to 3.0 mg, about 3.0 to 5.0 mg, about 5.0 to 7.0 mg, about 7.0 to 9.0 mg, or about 9.0 to 11.0 mg EZE per dose. In some preferred embodiments, the subject may be administered about 1 mg EZE per dose. In other preferred embodiments, the subject may be administered no more than about 10 mg EZE per dose. In a preferred embodiment, EZE may be administered to subject at least once. In another preferred embodiment, EZE may be administered to subject once or twice. In still other embodiments, EZE may be administered to subject for any number of times to obtain a reliable sample size.

[0041] It is to be understood that the present invention is not limited to the aforementioned doses. In preferred embodiments, the amount of EZE administered to the subject may be a sufficiently low amount such that EZE has minimal to no therapeutic effect, yet still allowing EZE and/or EZE-Gluc to be detectable by any appropriate system (e.g., LC, MS, LC/MS, immune assays) to indicate disease. In some preferred embodiments, the maximum amount of EZE administered to the subject may be an amount that does not have any toxic effects. In other preferred embodiments, the maximum amount of EZE administered to the subject may be a therapeutic dose. In yet other preferred embodiments, the amount of EZE administered to the subject may be any dose of EZE that allows for measurement of EZE-Gluc levels.

[0042] According to other embodiments, the present invention provides a method of monitoring progression of a hepatic disorder or disease in a subject over time. In one embodiment, the method comprises administering EZE to the subject, obtaining a non-biopsy, fluid sample (e.g., plasma and/or urine) after a period of time has passed from administering the EZE, measuring an amount of EZE and/or EZE-Gluc in the fluid sample, and comparing the amount of EZE and/or EZE-Gluc to a reference value in healthy patients without the disorder and without signs or symptoms of the disorder. In preferred embodiments, the plasma EZE-Gluc level in the subject is compared to the plasma EZE-Gluc level in the reference, the plasma EZE level in the subject is compared to the plasma EZE level in the reference, the urine EZE-Gluc level in the subject is compared to the urine EZE-Gluc level in the reference, and/or the urine EZE-Gluc level in the subject is compared to the urine EZE-Gluc level in the reference. In one embodiment, a deviation of the amount of EZE-Gluc from the reference value indicates a stage of the hepatic disease. In some embodiments, if the subject is below the reference threshold, the subject may not have NASH. In some embodiments, if the subject approaches the reference value, then they may be at risk for NASH. In some embodiments, if the subject surpasses the reference value, then the patient has NASH. In some embodiments, if the subject surpasses a higher reference value, then the patient has a more severe form of NASH As a non-limiting example, the reference threshold may be about 10 ng/ml of plasma depending on the dose, fluid being analysed, and time from administration. In some embodiments, the deviation in the amount of EZE and/or EZE-Gluc may be at least 50%, at least 100%, at least 200%, at least 300%, or at least 400% of the reference. In other embodiments, the amount of elevated EZE and/or EZE-Gluc is at least 10 times, 100 times, 1000 times, 10,000 times, or at least 100,000 times that of the reference. In some embodiments, the monitoring occurs at select times throughout the disorder, treatment, or patient care management comprising intervals of daily, weekly, monthly, and/or yearly.

[0043] According to further embodiments, the present invention provides a method of treating a hepatic disorder or disease. In some embodiments, the method comprises administering EZE to the subject, obtaining a non-biopsy, fluid sample after a period of time has passed from administering the EZE, measuring an amount of EZE and/or EZE-Gluc in the fluid sample, and comparing the amount of EZE and/or EZE-Gluc to a reference value in healthy patients without the disorder and without signs or symptoms of the disorder. In preferred embodiments, the plasma EZE-Gluc level in the subject is compared to the plasma EZE-Gluc level in the reference, the plasma EZE level in the subject is compared to the plasma EZE level in the reference, the urine level of EZE-Gluc level in the subject is compared to the urine EZE-Gluc level in the reference, and/or the urine EZE level in the subject is compared to the urine EZE level in the reference. Without wishing to limit the invention to a particular theory or mechanism, a deviation of the amount of EZE-Gluc from the reference value may be indicative of a hepatic disorder and the stage of the hepatic disease. In some embodiments, the amount of deviated EZE and/or EZE-Gluc levels is at least 50%, at least 100%, at least 200%, at least 300%, or at least 400% of the reference. Non-limiting examples comprise the plasma EZE-Gluc level in the subject being 200% higher compared to the plasma level of EZE-Gluc in the reference or the urine EZE-Gluc level in the subject being 100% higher compared to the urine level of EZE-Gluc in the reference. In other embodiments, the amount of elevated EZE and/or EZE-Gluc is at least 10 times, 100 times, 1000 times, 10,000 times, or at least 100,000 times that of the reference. A non-limiting example comprises the plasma EZE-Gluc level in the subject is 1000 times higher compared to the plasma level of EZE-Gluc in the reference.

[0044] In preferred embodiments, the method further comprises prescribing a therapy to the patient stage of the hepatic disease. In some embodiments, prescribing a therapy to the patient may involve one or more of dietary changes, exercise regimens, lifestyle changes, surgical interventions, and use of medications.

[0045] According to additional embodiments, the present invention features a method of monitoring the effectiveness of a treatment for a hepatic disorder or disease in a subject in need of such treatment. In some embodiments, the method comprises administering EZE to the subject prior to the initiation of the treatment (to serve as a baseline or initial assessment) and at any time following initiation of treatment (to serve as during treatment or on treatment assessments), obtaining a non-biopsy, fluid sample (e.g., plasma, urine) after a period of time has passed from administering the EZE, measuring an amount of EZE and/or ezetimibe-glucuronide (EZE-Gluc) in the fluid sample, comparing the amount of EZE and/or EZE-Gluc between the initial (pre-treatment) and post-treatment initiation (during treatment) values and to a reference value (e.g., compare plasma EZE-Gluc level in subject being monitored to the plasma EZE-Gluc level in reference), and monitoring the treatment to the subject based on the change of levels of EZE and/or EZE-Gluc from prior to initiation of therapy and between the subject and the reference, wherein the change indicates the efficacy of the treatment. Without wishing to limit the invention to a particular theory or mechanism, a deviation of the amount of EZE and/or EZE-Gluc from the reference value indicates a stage of the hepatic disease.

[0046] In preferred embodiments, the method comprises monitoring a treatment that is prescribed to the patient stage of the hepatic disease. In some embodiments, the subject can be identified as still having the hepatic disorder if the amount of EZE and/or EZE-Gluc in one or both of the plasma sample and the urine sample is elevated relative to the reference and/or to the baseline value (e.g., compare plasma EZE-Gluc levels in human subject to plasma EZE-Gluc levels in reference and/or to baseline levels prior to initiation of therapy; compare urine EZE-Gluc levels in human subject to urine EZE-Gluc levels in reference and/or baseline), possibly requiring a change in treatment. In some embodiments, if the subject has a level that is below the level obtained prior to initiating the treatment, the subject may be receiving benefit from the treatment. In some embodiments, if the level in a subject decreases and approaches the reference value, then the subject may be considered cured. In some embodiments, if the subject has a level that surpasses the initial value, then the patient continues to have the hepatic disorder. In some embodiments, the amount of elevated or deviated EZE and/or EZE-Gluc levels is at least 50%, at least 100%, at least 200%, at least 300%, or at least 400% of control (e.g., compare plasma EZE-Gluc in subject to plasma level of EZE-Gluc in the control). In other embodiments, the amount of elevated EZE and/or EZE-Gluc is at least 10 times, 100 times, 1000 times, 10,000 times 100,000 times that of the control (e.g., compare plasma EZE-Gluc in subject to plasma level of EZE-Gluc in the control). In other embodiments, the amount of elevated EZE and/or EZE-Gluc is compared to the patient's EZE and/or EZE-Gluc levels prior to initiation of therapy where any decrease (e.g., at least 1%) toward reference value would indicate improvement, abatement, or resolution of the hepatic disorder. In some embodiments, the decrease indicative of treatment efficacy is at least 1%, at least 5%, at least 10%, at least 25%, at least 50%, at 75%, or at least 100%.

[0047] In preferred embodiments, the time of EZE administration for biomarker testing prior to initiation of a treatment for a hepatic disorder comprises up to 0 day (e.g., same day as treatment initiation), up to 1 day, up to 2 days, up to 3 days, or up to 6 months prior to treatment initiation. In some embodiments, the time of EZE administration for biomarker testing prior to initiation of a treatment for a hepatic disorder may be any number of days appropriate to allow for the EZE measurement to be used in accordance with the present invention. In some embodiments, the treatment to the patient may change based on monitoring results and may involve one or more of dietary changes, exercise regimens, lifestyle changes, surgical interventions, and use of medications.

[0048] The present invention further features a method for delineating non-alcoholic steatohepatitis (NASH) from simple steatosis in a human subject. In some embodiments, the method comprises administering ezetimibe (EZE) to the human subject, determining an amount of EZE and/or ezetimibe-glucuronide (EZE-Gluc) in a plasma sample and/or a urine sample obtained from the human subject after some time the human subject was administered ezetimibe (EZE), comparing the amount of EZE and/or EZE-Gluc in one or both of the plasma sample and the urine sample to a reference value, wherein the reference is from subjects with steatosis, and identifying the human subject as having NASH if the amount of EZE and/or EZE-Gluc in one or both of the plasma sample and the urine sample is elevated relative to the steatosis reference. In some embodiments, the amount of elevated or deviated EZE and/or EZE-Gluc levels is at least 50%, at least 100%, at least 200%, at least 300%, or at least 400% of steatosis (e.g., compare plasma EZE-Gluc in subject to plasma level of EZE-Gluc in the steatosis reference). In other embodiments, the amount of elevated EZE and/or EZE-Gluc is at least 10 times, 100 times, 1000 times, 10,000 times 100,000 times that of the steatosis (e.g., compare plasma EZE-Gluc in subject to plasma level of EZE-Gluc in the steatosis reference).

[0049] The present invention also features a method for determining the efficacy of a therapeutic treatment for NASH in patients in need of such therapy. In some embodiments, the method comprises administering EZE to the human subject prior to initiation of treatment for NASH (to serve as initial or baseline assessment) and at any time following initiation of NASH treatment, determining an amount of EZE and/or EZE-Gluc in a plasma sample and/or a urine sample obtained from the human subject after some time the human subject was administered EZE, comparing the amount of EZE and/or EZE-Gluc in one or both of the plasma sample and the urine sample to the initial EZE and/or EZE-Gluc in one or both of the plasma sample and the urine sample of the patient prior to initiation of therapy, and determining the efficacy of the treatment if the amount of EZE and/or EZE-Gluc in one or both of the plasma sample and the urine sample has decreased over time relative to the initial test prior to initiation of treatment, wherein any decrease over time (e.g., at least 1%) indicates that the treatment is efficacious. In some embodiments, the decrease indicative of treatment efficacy is at least 1%, at least 5%, at least 10%, at least 25%, at least 50%, at 75%, or at least 100%.

[0050] In some embodiments, the time of EZE administration for biomarker testing prior to initiation of a treatment for a hepatic disorder comprises up to 0 day (e.g., same day as treatment initiation), up to 1 day, up to 2 days, up to 3 days, or up to 6 months prior to treatment. In some embodiments, the time of EZE administration for biomarker testing prior to initiation of a treatment for a hepatic disorder may be any number of days appropriate to allow for the EZE measurement to be used in accordance with the present invention. In some embodiments, the treatment to the patient may change based on monitoring results and may involve one or more of dietary changes, exercise regimens, lifestyle changes, surgical interventions, and use of medications.

[0051] Consistent with previous embodiments, other markers may be used that can distinguish NAFL from NASH, alternatively or in conjunction with EZE-Gluc. Non-limiting examples of these other biomarkers include CK-18 as well as perillipin-1 (PLIN-1) and PLIN-2 lipid droplet proteins, age, weight, ALT, AST, triglyceride, diabetes, TIMP-1, hepascore, hyaluronic acid, elastography, acoustic radiation, fibroscan, MRI, CT, or PET scans.

EXAMPLES

[0052] The following is a non-limiting example of the present invention. It is to be understood that said example is not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.

Example 1: EZE-GLUC Increased Plasma Retention and Decreased Biliary Elimination in a NASH Animal Model

[0053] Ezetimibe was orally administered to control and MCD rats. Blood and bile were collected and EZE and ZE-Guc were measured by LC-MS/MS. FIG. 2A shows the amount of ezetimibe (EZE) and glucuronidated ezetimibe (EZE-Gluc) in plasma and bile after the oral administration of EZE to control and MCD (NASH) rats. The solid and open arrows indicate where NASH caused an increase and decrease, respectively, in the amount of compound in the biological fluid indicated.

[0054] Ezetimibe was intravenously administered to control and MCD rats. Blood and bile were collected and EZE and EZE-Guc were measured by LC-MS/MS. FIG. 2B shows the amount of ezetimibe (EZE) and glucuronidated ezetimibe (EZE-Gluc) in urine after the intravenous administration of EZE to control and MCD (NASH) rats. The solid arrows indicate where NASH caused an increase in the amount of compound in the biological fluid indicated.

Example 2: Method of EZE/EZE-Gluc Assessment in Human Subjects

[0055] A subject at risk for a hepatic disorder arrives at a testing site on the morning of the assessment after an overnight fast who was previously advised to avoid any product containing either APAP or EZE (including TYLENOL, NyQuil, Robitussin, Zetia etc.) for at least three days prior to the assessment. Following baseline blood (20 cc) and urine collection, the subject receives one standard oral dose of EZE (e.g., 10 mg). Subjects are allowed access to food and water during the 6 h assessment. Blood is collected at 0.25, 0.5, 1, 1.5, 2, 3, 4, and 6 h post drug administration by an appropriately trained professional (e.g., attending nursing staff). Approximately 7 cc of blood is collected into a heparinized vacutainer tube at each time point, and the samples are centrifuged within 30 min of blood collection. Due to the effects of patient hydration and kidney health on the variability in urine concentration, urine levels of metabolites are calculated as a total amount rather than as a concentration. Urine is collected at 1, 2, 4 and 6 h, the total volume is noted, and a small (10 mL) sample is reserved for LC-MS/MS analysis.

[0056] In some embodiments, biochemical analysis is performed on pre-dose blood samples, and measure alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GT), total cholesterol and fractions, triglycerides, fasting plasma glucose, and insulin. Height and weight measurements will also be recorded for BMI calculation and statistical analysis. Cytokeratin-18 levels will be determined using a commercially available ELISA kit.

[0057] Using the collected blood and urine samples, ezetimibe and metabolite quantification can be performed according to standard procedures. Briefly, tolbutamide is used as an internal standard, and samples are extracted using Oasis HLB solid phase extraction. EZE and EZE-Gluc are measured according to standard analytical procedures (20,58). For example, the LC-MS/MS system is an Acquity Ultra Performance Liquid Chromatography (UPLC) system with an auto-sampler (Waters, Milford, Mass.) coupled with a Waters Micromass Quattro Premier XE tandem mass spectrometer (Waters) equipped with MassLynx4.1 software (Waters). The chromatography consists of a gradient beginning at 20:80 (v/v) acetonitrile-water and ending at 80:20 (v/v) acetonitrile-water for 7 min with a flow rate of 0.25 mL/min on an Acquity UPLC BEH C18 column (1.7 m; 2.150 mm; Waters). Electrospray ionization in the negative ion mode is used, and the analytes are detected using a multiple reaction monitoring (MRM) method. The precursor and product ions (m/z) are 408 and 271, respectively, for EZE, 584 and 271, respectively, for EZE-Gluc, and 269 to 170, respectively, for the internal standard.

[0058] The levels of EZE and/or EZE-Gluc are then analyzed and compared (e.g., EZE to EZE levels; EZE-Gluc to EZE-Gluc levels) to a control reference value obtained from healthy individuals without a hepatic disorder, without signs/symptoms of a hepatic disorder, or without a risk for a hepatic disorder. For example, average peak blood concentrations of 92 ng/mL and 4.5 ng/mL for EZE-Gluc and EZE, respectively, were found in normal healthy individuals after a 10 mg oral dose of EZE. In some embodiments, the subjects with NASH or at risk of NASH have levels of EZE-Gluc at least 50%, at least 100%, at least 200%, at least 300%, or at least 400% higher than the control at early time points, for example one hour in blood or two hours in urine. At later timepoints (e.g., after 2 hours in the blood; 4 hours in the urine), the levels become much more elevated, as much as 10-100,000 times as compared to the control. In some embodiments, the elevation of EZE-Gluc will only be present at the stage of NASH. In other embodiments, EZE-Gluc can be used to differentiate between grades of NASH. In certain embodiments, the levels of EZE-Gluc in subjects with steatosis will be no different than control.

[0059] As used herein, the term about refers to plus or minus 10% of the referenced number. Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in the present application is incorporated herein by reference in its entirety.

[0060] Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase comprising includes embodiments that could be described as consisting essentially of or consisting of, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase consisting essentially of or consisting of is met.