A simplified method for measuring liver function

Abstract

A simplified method for determining liver function is provided based on a compartmental model (CM) approach for the analysis of various liver indices, including the cholate SHUNT test, which simultaneously and closely approximates and elucidates the fundamental physiology of hepatic uptake of cholate from the systemic and portal circulations.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application was filed as a PCT international patent application on November 2, 2023, and claims the benefit of and priority to U.S. Provisional Patent Application Nos. 63 / 422,337, filed November 3, 2022, 63 / 505,880, filed June 2, 2023, and 63 / 581,496, filed September 8, 2023, each of which is incorporated by reference herein in its entirety. [Background technology]

[0002] Background of the Invention Chronic liver disease (CLD) affects millions of people worldwide. Estimates suggest that as many as 50 million Americans have CLD, over 15 million have fibrosis, and over 4 million have cirrhosis. Common etiologies of CLD include alcoholic liver disease, chronic viral hepatitis B or C, autoimmune hepatitis, primary biliary cholangitis, primary sclerosing cholangitis, and genetic disorders. However, as a result of the obesity epidemic, the most common emerging and expanding etiology for CLD is metabolic dysfunction-associated fatty liver disease (MASLD), formerly known as nonalcoholic fatty liver disease (NAFLD), or metabolic dysfunction-associated steatohepatitis (MASH), formerly known as nonalcoholic steatohepatitis (NASH). The Cholate SHUNT Liver Function Test quantifies liver health (DSI, Disease Severity Index) to aid healthcare providers, patients, and payers in the management of CLD patients.

[0003] The cholate SHUNT test quantifies liver function from portal and systemic clearance using intravenous carbon-13-labeled cholate (13C-CA) and oral stable, non-radioactive deuterium-labeled cholate (d4-CA) simultaneously. The test is minimally invasive, well tolerated, and requires only peripheral venous blood sampling. The Disease Severity Index (DSI) score, a single score of liver health ranging from 0 (healthy) to 50 (severely impaired), has demonstrated reliability in reproducibility studies (Burton JR, Helmke S, Lauriski S, Kittelson J, Everson GT. The within-individual reproducibility of the disease severity index from the HepQuant SHUNT test of liver function and physiology. Transl Res. 2021;233:5-15), and has been used to detect early disease, quantify disease severity, monitor disease progression, and measure treatment efficacy (Helmke S, Colmenero J, Everson GT. Noninvasive assessment of liver function. Curr Opin Gastroenterol. 2015;31). The test uniquely enables pharmaceutical companies to gauge the effectiveness of their therapeutic agents by measuring improvements in liver function without waiting for clinically observed progressive liver failure. A significant study was recently completed linking SHUNT trial results to the likelihood of large esophageal varices, a precursor to variceal bleeding and the most lethal complication of CLD (ClinicalTrials.gov. The SHUNT-V Study for Varices NCT03583996).

[0004] The cholate SHUNT test of liver function and physiology can be useful for monitoring treatment efficacy and predicting risk to clinical outcomes. The HepQuant Cholate SHUNT test quantifies liver dysfunction from the simultaneous clearance of cholate from the systemic and portal circulations for the purpose of monitoring treatment efficacy or predicting risk to clinical outcomes. Test parameters derived from a non-compartmental minimal model (MM) are reproducible and reliable (Translational Research 2021).

[0005] U.S. Pat. No. 8,613,904, Everson et al., discloses a cholate SHUNT test method for assessing liver function in patients, which involves obtaining patient serum samples after administration of two distinguishable stable isotope-labeled cholate compounds, laborious sample processing, and analysis of the patient serum samples using GC-MS.

[0006] U.S. Patent No. 8,778,299, Everson, discloses a cholate SHUNT test method for assessing liver function, which involves obtaining, processing, and analyzing a patient's serum sample using HPLC-MS after administration of two distinguishable stable isotope-labeled cholate compounds. A method for determining portal vein hepatic filtration rate (portal HFR, FLOW) from oral administration of distinguishable drugs is also provided.

[0007] U.S. Patent No. 9,091,701, Everson, discloses a method for determining liver function in a patient and obtaining a Disease Severity Index (DSI) value, comprising obtaining, processing, and analyzing a patient serum sample using HPLC-MS after administration of two distinguishable stable isotope-labeled cholate compounds.

[0008] No. 8,961,925, Everson, discloses a method for conducting a STAT test involving oral administration of an identifiable drug and subsequent measurement of the identifiable compound in a single blood or serum sample using HLPC-MS. A method for estimating portal vein hepatic filtration rate (portal HFR) from STAT values ​​is also provided.

[0009] U.S. Patent Application Publication No. 2021 / 0318274(A1), Everson et al., discloses improved methods for blood or serum sample preparation, analyte detection and quantification that can be applied to one or more of the SHUNT, FLOW, STAT, and DSI liver function tests.

[0010] The cholate SHUNT liver function test involves an intravenous (IV) dose of carbon-13 labeled cholate (13C-CA), a simultaneous oral dose of deuterium labeled cholate (d4-CA), and five peripheral venous blood draws over 90 minutes to simultaneously measure portal and systemic clearance. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] U.S. Patent No. 8,613,904 [Patent Document 2] U.S. Patent No. 8,778,299 [Patent Document 3] U.S. Patent No. 9,091,701 [Patent Document 4] U.S. Patent No. 8,961,925 [Patent Document 5] U.S. Patent Application Publication No. 2021 / 0318274(A1) [Patent Document 6] U.S. Patent No. 9,639,665 [Patent Document 7] U.S. Patent No. 9,417,230 [Patent Document 8] U.S. Patent No. 10,215,746 [Patent Document 9] WO 2021 / 207683 A1 [Patent Document 10] U.S. Patent No. 10,222,366 [Patent Document 11] U.S. Patent No. 9,759,731 [Patent Document 12] U.S. Patent No. 10,520,517

Non-licensed literature

[0012] [Non-licensed document 1] Burton JR, Helmke S, Lauriski S, Kittelson J, Everson GT. The within-individual reproducibility of the disease severity index from the HepQuant SHUNT test of liver function and physiology. Transl Res. 2021;233:5~15 pages [Non-licensed document 2] Helmke S, Colmenero J, Everson GT. Noninvasive assessment of liver function. Curr Opin Gastroenterol. 2015;31 [Non-licensed document 3] ClinicalTrials.gov.The SHUNT-V Study for Varices. clinicaltrials.gov / ct2 / show / NCT03583996.July 20, 2022アクセス

Non-licensed Document 4

Non-licensed Document 5

[0013] A simplified test that provides a reliable measurement of liver function, significantly reducing the time and invasiveness of the test, is desirable. [Means for solving the problem]

[0014] SUMMARY OF THE INVENTION A simplified liver function test is provided that uses naturally occurring endogenous cholate labeled with a stable isotope to probe hepatocyte uptake and hepatic blood flow, assessing both liver function and physiology. The results provide accurate information about liver damage in individuals at risk for liver disease / with liver disease, to monitor treatment efficacy, and to determine risk for clinical complications.

[0015] The present disclosure provides a series of second-generation liver function tests that provide accurate measures of liver function that correlate with first-generation liver function tests (STAT, FLOW, and SHUNT tests) but are significantly simplified in terms of test administration.

[0016] Liver function test parameters (DSI, HFR, SHUNT, HR, etc.) were measured by oral and IV discriminative cholate clearance area under the curve (AUC Oral , AUC IV ) requires accurate and reliable measurement of hepatocellular function and portal circulation. The SHUNT test assesses both hepatocellular function and portal circulation, making it a comprehensive diagnostic tool to address the pathogenesis of all types of CLD. However, the SHUNT test (SHUNT V1.0) requires both oral and intravenous (IV) cholate doses and six peripheral venous blood samples collected over a 90-minute period. Potential sources of error associated with IV infusion and multiple blood samples include poor IV access (difficulty in locating the vessel or removing the catheter from the vessel), extravasation, errors in sampling timing, and skill variability in test administration.

[0017] Provided herein are simplified liver function tests, including the Cholate DuO and SHUNT V2.0 liver function tests, that reduce the number of blood samples by two-thirds and test time by one-third compared to SHUNT V1.0, provide highly reproducible measurements of liver function and physiology, and have the potential to improve prediction of clinical outcomes, enhance patient monitoring, and inform decisions regarding expensive drugs, reagents, and procedures.

[0018] The present disclosure provides simplified Duo and TRIO liver function tests that require only a limited number of data points (e.g., only two oral data points for Duo, no more than two oral data points for Duo, or two oral plus one or two IV data points for TRIO), and specialized analytical techniques for fitting systemic and portal clearance curves therefrom. Analytical methodologies are provided herein for each test.

[0019] The present disclosure provides a method for assessing liver function in a subject having or suspected of having or suffering from liver disease, comprising the steps of: obtaining blood or serum sample concentration data of orally administered distinguishable cholate compounds taken from the subject at two time points after oral administration; measuring the area under the curve (AUCoral) of the blood or serum concentration of orally administered distinguishable cholate compounds in the subject, comprising: obtaining blood or serum sample concentration data of orally administered distinguishable cholate compounds taken from the subject at two time points after oral administration; simulating a full oral clearance curve using a compartmental model of oral cholate clearance, the compartmental model including the oral distinguishable cholate compound concentration data at the two time points, body mass index (BMI), body weight (BW), and hematocrit (Hct) input values ​​in the subject, and calculating an area involving trapezoidal numerical integration to obtain the AUCoral; and calculating one or more indices of liver disease in the subject using the AUCoral, wherein the one or more indices are related to liver function in the subject.

[0020] A method for obtaining data at first and second time points after oral administration of a distinguishable cholate compound can include receiving first and second blood or serum samples from a subject at first and second time points after a single oral dose of a first distinguishable cholate compound; and analyzing the samples to obtain oral concentration data at the first and second time points, where optionally, the blood or serum samples are obtained within about 180 minutes, 120 minutes, 90 minutes, or about 75 minutes after oral administration. In some cases, the first and second blood or serum samples are obtained from the subject at least about 5 minutes to about 75 minutes, 10 minutes to 70 minutes, 20 minutes to 60 minutes, 25 minutes to 55 minutes, 30 minutes to 50 minutes, 35 minutes to 45 minutes, or about 40 minutes apart. In some cases, the first and second blood or serum samples are obtained from the subject about 10 to 30 minutes and about 40 to 80 minutes after oral administration, respectively. In some instances, the first and second blood or serum samples were collected from the subject about 15-25 minutes and about 50-70 minutes, respectively, after oral administration. In some instances, the first and second blood or serum samples were collected from the subject about 20 minutes and about 60 minutes, respectively, after oral administration.

[0021] A method for obtaining data at first and second time points after oral administration of a distinguishable cholate compound can include receiving a single blood or serum sample from a subject after administration of a first oral dose of a first distinguishable cholate compound and a second oral dose of a second distinguishable cholate compound to the subject. The method can include analyzing the single sample to obtain oral concentration data for the first distinguishable cholate compound and the second distinguishable cholate compound at two time points. The single blood or serum sample was obtained from the subject within about 180 minutes, 120 minutes, 90 minutes, or about 75 minutes after administration of the first oral dose. In some cases, the first and second oral doses were administered to the subject at least about 5 minutes to about 75 minutes, 10 minutes to 70 minutes, 20 minutes to 60 minutes, 25 minutes to 55 minutes, 30 minutes to 50 minutes, 35 minutes to 45 minutes, or about 40 minutes apart. In some cases, a single sample was simultaneously collected from the subject about 10-30 minutes after the second oral dose and about 40-80 minutes after the first oral dose to obtain data at the first and second time points. In some cases, a single sample was simultaneously collected from the subject about 15-25 minutes after the second oral dose and about 55-65 minutes after the first oral dose to obtain data at the first and second time points. In some cases, a single sample was simultaneously collected from the subject about 20 minutes after the second oral dose and about 60 minutes after the first oral dose to obtain data at the first and second time points.

[0022] In some embodiments, a method for assessing liver function in a subject suspected of having or suffering from liver disease can include estimating the area under the curve (AUCiv) of the blood or serum concentration of an intravenously administered distinguishable cholate compound, comprising a linear regression model. The AUCiv can be estimated from the linear regression model according to Equation 11A: AUC IV =β0+β BW BW+β PO,20 C PO,20 +β PO,60 C PO,60 +β HFR,P HFR P ,Equation 11A

[0023] where β is the intercept coefficient, optionally 161.972; BW is the body weight coefficient, optionally the body weight coefficient is 0.6459; BW is the subject's body weight in kg; β PO,20 is the orally administered discriminant cholate concentration coefficient at the first time point, and optionally β PO,20 is 16.9249;C PO,20 is the orally administered discernible cholate concentration at the first time point; β PO,60 is the orally administered discriminant cholate concentration coefficient at the second time point, and optionally β PO,60 is 89.2405;C PO,60 is the orally administered discernible cholate concentration at the second time point; β HFR,P is the portal vein HFR coefficient, and optionally the portal vein HFR coefficient is -0.4755; HFR P is the subject's measured portal vein HFR.

[0024] In some embodiments, a method for assessing liver function in a subject having or suspected of having or suffering from liver disease can include obtaining blood or serum sample concentration data of an intravenously administered third distinguishable cholate compound at one of two time points after oral administration, and estimating the area under the curve (AUC) of the blood or serum concentration of the intravenously administered distinguishable cholate compound, comprising exponentially fitting the intravenous concentration data to a systemic cholate clearance curve including a rapid phase, a moderate phase, and a slow phase of clearance over a period of at least about 180 minutes after i.v. administration of the intravenous dose. In some cases, the third distinguishable cholate was intravenously administered at least about 5 to about 75 minutes, 10 to 70 minutes, 20 to 60 minutes, 25 to 55 minutes, 30 to 50 minutes, 35 to 45 minutes, or about 40 minutes after the first oral dose. In some cases, the blood or serum sample collected at a single time point was collected within about 90 minutes, 75 minutes, 60 minutes, 45 minutes, 30 minutes, or about 20 minutes after intravenous administration.

[0025] An exponential fit to the rapid phase (Y) of the systemic cholate clearance curve can be calculated according to Equation 20:

number

number

number

number

[0026] An exponential fit to the rapid phase (Y1) of the systemic cholate clearance curve can be calculated according to Equation 21:

number

[0027] An exponential fit to the slow phase (Y2) of the systemic cholate clearance curve can be calculated according to Equation 22:

number

[0028] The area under each of the three exponential curve fits for the fast, moderate, and slow phases was calculated by trapezoidal numerical integration, and the AUC IV can be summed to estimate

[0029] A simplified compartment model is provided for calculating AUCoral in a subject from the provided orally administered distinguishable cholate compound concentration data at two time points. The simplified compartment model of oral clearance can include estimating compartment volumes of multiple compartments in the subject and flow parameters between multiple compartments in the subject. The simplified compartment model of oral cholate clearance can further include estimating cholate binding and dose administration in the subject.

[0030] The multiple compartments in the subject can include at least the systemic, portal, and hepatic compartments.

[0031] The step of estimating the compartment volumes of the plurality of compartments includes estimating the total body compartment volume (V S ), portal vein compartment volume (V P ), and liver compartment volume (V L ), optionally with each compartment volume in liters (L).

[0032] Whole body compartment volume (V S ) can be estimated according to Equation 4A: V S =TBV·(1-Hct) Equation 4A During the ceremony, TBV is the total blood volume in the subject according to equation 4:

number

[0033] Portal compartment volume (V P ) can be estimated according to equation 4B: V P =0.25 V S Equation 4B

[0034] Liver compartment volume (V L ) can be estimated according to Equation 5A: V L =(0.275 22.46 BW d L f plasma ) / 1000 Equation 5A In the formula, d L is 1.06 g mL -1 is the hypothetical liver tissue density.

[0035] A simplified compartmental model can involve the estimation of flow parameters between multiple compartments involving a system of first-order ordinary differential equations 1A-3:

number

number

number

[0036] where V is the total body (V S ), portal vein (V P ), and liver (V L ) is the volume of each of the compartments, and C is the total body volume (C S ), portal vein (C P ), and liver (C L ) is the concentration of cholate in each of the compartments, q is the flow rate between the compartments, and Cl H is hepatic clearance.

[0037] The simplified compartment model is based on the total hepatic inflow (Q L ), splanchnic arterial circulation (q SP ), hepatic portal vein inflow to the liver (q PL ), total hepatic venous return to the systemic circulation (q LS ), and hepatic arterial inflow to the liver (q SL ) can be included in estimating

[0038] Total hepatic inflow to the liver (Q L ) is calculated by estimating the hepatic artery flow to the liver (q SL ) and portal vein (q PL ) Inflows can include both: Q L =q SL +q PL ,(L minutes -1 ) Equation 6A In the formula, q SL is the hepatic arterial inflow velocity to the liver, and q SL =0.25 Q L (L minutes -1 ) and q PL is the portal inflow rate to the liver, and q PL =q SP (L minutes -1 ) and ;q SP is the splanchnic artery blood flow velocity to the abdominal intestinal organs, and q SP =0.75 Q L,init (L minutes -1 ) and optionally Q L An initial estimate for is approximately 1 L min -1 kg -1 Liver wet mass.

[0039] Hepatic clearance (Cl H ) can be estimated by Equation 7A: Cl H =Q L ·ER Equation 7A where ER is the extraction ratio in the subject and is assumed to be 0.7 for cholate.

[0040] In some embodiments, the method includes the steps of: obtaining subject-derived input data including blood or serum sample concentration data of an orally administered distinguishable cholate compound taken from the subject at two time points within 180 minutes after oral administration, a body mass index (BMI), optionally a hematocrit (Hct), and an estimated volume of distribution (Vd) for the subject; fitting the input data to a trained function-fitting neural network comprising a learning algorithm to generate a plurality of output points for oral and intravenous distinguishable cholate clearance curves; constructing oral and IV distinguishable cholate clearance curves from the fitted data; and calculating an AUC for the subject comprising trapezoidal numerical integration. Oral Value and AUC IV measuring the AUCoral value and / or AUC IV

[0009] Methods for assessing liver function in a subject having or suspected of having or suffering from liver disease are provided, comprising using the values ​​of Hct and Hct+H ...

[0041] In some embodiments, the method includes obtaining input data derived from a subject, the input data including concentration data of a blood or serum sample of an orally administered first distinguishable cholate compound taken from the subject at two time points within 180 minutes after oral administration, concentration data of a blood or serum sample of an intravenously administered second distinguishable cholate compound taken from the subject at one time point within 180 minutes after intravenous administration, an estimated volume of distribution (Vd) in the subject, and an estimated initial intravenous distinguishable cholate concentration at 0 minutes of the intravenously administered second distinguishable cholate based on Vd; fitting the input data to a trained function-fitting neural network comprising a learning algorithm to generate a plurality of output points for oral and intravenous distinguishable cholate clearance curves; constructing oral and IV distinguishable cholate clearance curves from the fitted data; and calculating an AUC for the subject comprising trapezoidal numerical integration. Oral Value and AUC IV measuring the AUCoral value and / or AUC IV

[0013] In some embodiments, an estimated Vd (L per kg body weight) in a subject can be estimated by Equation 16A:

number

[0042] The neural network can be configured for regression tasks and includes a two-layer feedforward network that includes a hidden layer and an output layer, optionally including a sigmoidal transformation function in the hidden layer and a linear transformation function in the output layer.

[0043] The neural network can include a learning algorithm trained on a training dataset including multiple distinguishable oral and intravenous cholate clearance curves estimated by a non-compartmental minimal model (MM) from a combination of normal control subjects and patients with chronic liver disease. The learning algorithm can be selected from the group consisting of Levenberg-Marquardt backpropagation, Bayesian regularization, BFGS quasi-Newton method, resilient backpropagation, scaled conjugate gradient, conjugate gradient with Powell / Beale restart, Fletcher-Power conjugate gradient, Polak-Ribiere conjugate gradient, one-step secant method, variable learning rate gradient descent, gradient descent with momentum, and gradient descent learning algorithms.

[0044] In some embodiments, the output points for the distinguishable oral and intravenous cholate clearance curves include 5 minute increments from 0 to 180 minutes after oral administration, resulting in 37 time points for each of the oral and IV clearance curves.

[0045] In some cases, the initial intravenous discernible cholate compound concentration at 0 minutes is calculated using Equation 16A:

number

[0046] (a) administering a dose of a first distinguishable cholate compound to a subject; oral ) and administering to the subject a dose of a second distinguishable cholate compound. iv receiving a plurality of blood or serum samples taken from the subject following simultaneous intravenous co-administration of (b) quantifying the concentrations of the first and second distinguishable cholate compounds; and (c) generating individual subject oral and intravenous clearance curves from the concentrations of the first and second distinguishable cholate compounds, including using computer algorithm curve fitting to model the oral and intravenous clearance curves; and calculating the individualized areas under the oral and intravenous clearance curves (AUCoral) and (AUCiv) for the subject, respectively, wherein the multiple samples include blood or serum samples taken from the subject at at least five time points; and The construction of individual intravenous clearance curves is Equation 16A:

number

[0013] Provided is a method for assessing liver function in a subject having or suspected of having or suffering from liver disease, comprising:

[0047] One or more indices of liver disease were assessed in the subjects using portal vein hepatic filtration fraction (HFRp), total body hepatic filtration fraction (HFRs), cholate SHUNT, liver disease severity index (DSI), and indexed hepatic reserve (HR indexed ), and algebraic hepatic reserve (HR algebraic ) can be selected from the group consisting of

[0048] The portal hepatic filtration rate (HFRp) in a subject can be calculated by Equation 10A:

number

[0049] Systemic hepatic filtration rates (HFRs) can be calculated according to Equation 12:

number

[0050] Cholate SHUNT(F) can be calculated according to Equation 13:

number

[0051] The Liver Disease Severity Index (DSI) can be calculated according to Equation 14:

number

[0052] Hepatic reserve (HR) can be calculated according to equation 15:

number

[0053] The disclosed methods can include comparing one or more indices of liver disease in a subject to one or more cutoff values ​​as indicators of relative liver function in the subject. The cutoff values ​​can be derived from one or more normal healthy controls, a group of known patients, or within subjects over time.

[0054] The known patient group can be suffering from any disease or condition.Disease or condition can be selected from the group consisting of: chronic liver disease with fibrosis stage; portal hypertension; Childs-Turcotte-Pugh (CTP) score A; CTP score B; CTP score C; Model for End-Stage Liver Disease (MELD) progression score; primary sclerosing cholangitis (PSC) not listed for transplantation; PSC listed for liver transplantation; PSC listed for liver transplantation without varices; PSC listed for liver transplantation with varices; ascites; stoma bleeding; splenomegaly; varices; large varices, variceal bleeding; hepatic encephalopathy, decompensation; or liver disease-related death.

[0055] Fibrosis stage can be determined by methods including liver biopsy or elastography. Liver biopsy can be used to determine an Ishak fibrosis score (liver biopsy) of F2 (mild portal fibrosis), F3, F4 (moderate bridging fibrosis), F5 (nodular and incomplete cirrhosis), or F6 (cirrhosis).

[0056] The one or more indices of liver disease can be utilized for a purpose selected from the group consisting of determining the need for treatment, predicting response to treatment, monitoring the effectiveness of treatment, and predicting the risk of a clinical outcome in a subject.

[0057] In some embodiments, the one or more indices of liver disease can be utilized for a purpose selected from the group consisting of determining the need for treatment, predicting response to treatment, monitoring the effectiveness of treatment, predicting large esophageal varices, individualized dosing of one or more drugs, and predicting the risk of a clinical outcome in a subject. The one or more indices of liver disease can be used for one or more precision medicine applications, such as individualized dosing of one or more drugs related or unrelated to the treatment of liver disease based on the estimated oral bioavailability of the drug.

[0058] The methods of the present disclosure can include providing one or more indices of liver disease to a medical professional for purposes of formulating a treatment plan in the subject. The subject can be a human subject.

[0059] The liver disease can be any known chronic liver disease (CLD). The liver disease can be selected from the group consisting of chronic hepatitis C (CHC), chronic hepatitis B, metabolic dysfunction-related alcoholic liver disease (Met-ALD), alcohol-related liver disease (ALD), fatty liver disease (SLD), also known as fatty liver disease, alcoholic steatohepatitis (ASH), alcoholic hepatitis (AH), metabolic dysfunction-related fatty liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), steatosis, metabolic dysfunction-related steatohepatitis (MASH), formerly known as non-alcoholic steatohepatitis (NASH), autoimmune liver disease, cryptogenic cirrhosis, hemochromatosis, Wilson's disease, alpha-1-antitrypsin deficiency, liver cancer, liver failure, cirrhosis, primary sclerosing cholangitis (PSC), and other cholestatic liver diseases.

[0060] Monitoring the need for treatment in a subject can include determining one or more indices of liver disease in the subject; and comparing the one or more indices of liver disease to one or more cutoff values, wherein a change in the one or more indices of liver disease compared to the cutoff values ​​is indicative of the need for treatment in the subject.

[0061] The treatment for chronic liver disease can be selected from the group consisting of antiviral therapy, antifibrotic therapy, antibiotics, immunosuppressive therapy, anticancer therapy, ursodeoxycholic acid, farnesoid X receptor ligands, insulin sensitizers, interventional therapy, liver transplantation, lifestyle modification, dietary restriction, low glycemic index diet, antioxidants, vitamin supplements, transjugular intrahepatic portosystemic shunting (TIPS), catheter-based thrombolysis, balloon dilation and stent placement, balloon dilation and drainage, weight loss, exercise, and alcohol avoidance. The first distinguishable cholate compound can be a first stable isotope-labeled cholate compound. The second distinguishable cholate compound can be a second stable isotope-labeled cholate compound. The third distinguishable cholate compound can be a third stable isotope-labeled cholate compound. BRIEF DESCRIPTION OF THE DRAWINGS [Brief explanation of the drawings]

[0062] [Figure 1A]

[0023] Figure 1 shows a schematic diagram illustrating the DuO cholate liver function test v1.0 parameters for estimating portal vein clearance using one oral cholate dose and two blood draws (DuO v1.0) to obtain two orally administered, distinguishable cholate (e.g., d4-CA) concentration time points. Both versions of the DuO test include the same analysis method. [Figure 1B]

[0023] Figure 1 shows a schematic diagram illustrating the DuO Cholate Liver Function Test v2.0 parameters for estimating portal vein clearance using two distinct oral cholate doses and one blood draw (DuO v2.0) to obtain two distinct orally administered cholate (e.g., d4-CA, 13C-CA) concentration time points. Both versions of the DuO test include the same analysis method. [Figure 2A] FIG. 1 shows a schematic diagram illustrating the TRIO Cholate Liver Function Test v1.0 (TRIO v1.0), which estimates both portal and systemic cholate clearance using one oral dose + one IV dose + two blood draws. Both versions of the TRIO test use the same analytical method. [Figure 2B] FIG. 1 shows a schematic diagram illustrating the TRIO Cholate Liver Function Test v2.0 (TRIO v2.0), which estimates both portal and systemic cholate clearance using two oral doses + one IV dose + one blood draw. Both versions of the TRIO test use the same analytical method. [Figure 3] Figure 1 shows an exemplary graph of TRIO cholate liver function test exponential fitting to the systemic clearance curve of intravenously administered identifiable cholate (C-CA) compared to the minimal model (MM). This non-compartmental analysis method involves exponential fitting of systemic cholate clearance using only a single IV time point (e.g., 20-minute C-CA concentration). In TRIO 1.0, a second sample at 60 minutes is also analyzed. The exponential fitting divides the curve into three sections or phases: fast, moderate, and slow (0-20 minutes, 20-45 minutes, and 45-180 minutes for Y0, Y1, and Y2, respectively). [Figure 4]Figure 1 shows the AI ​​DuO and AI TRIO neural network architectures. The neural network outputs selected points from the oral and IV curves estimated by the MM analysis method (i.e., SHUNT V1.0, 5-minute increments from 0 to 180 minutes, resulting in 37 time points for both oral and IV curves, which are rearranged into a 74 x 1 array). The network is configured for regression tasks and includes a two-layer feedforward network with a sigmoidal transformation function in the hidden layer and a linear transformation function in the output layer. The hidden layer size was optimized with respect to performance (mean squared error) on the test set. [Figure 5A] Figure 1 shows six graphs illustrating the correlation of portal vein HFR (HFRP) among second-generation cholate tests (DuO, TRIO, AI DuO, AI TRIO) and first-generation SHUNT test results analyzed by the MM (upper panel) and MMVd methods (lower panel). The TRIO graph is the same as that for DuO and is not shown. [Figure 5B] FIG. 1 shows Table 1, which contains HFRP reproducibility by analytical method as measured by coefficient of variation (CV) and intraclass correlation coefficient (ICC) for patient groups (control, N=16; NASH, N=16; HCV, N=16). [Figure 6A] FIG. 1 shows eight graphs illustrating the correlation of whole-body HFR (HFRS) among second-generation cholate tests (DuO, TRIO, AI DuO, AI TRIO) and first-generation SHUNT test results analyzed by the MM and MMVd methods. [Figure 6B] FIG. 6 shows Table 6, which contains HFRS reproducibility by analytical method as measured by coefficient of variation (CV) and intraclass correlation coefficient (ICC) for patient groups including NASH and HCV (control, N=16; NASH, N=16; HCV, N=16). [Figure 7A] FIG. 1 shows eight graphs illustrating the correlation of SHUNT% among second generation cholate tests (DuO, TRIO, AI DuO, AI TRIO) and first generation HepQuant SHUNT test results analyzed by the MM and MMVd methods. [Figure 7B] FIG. 8 shows Table 8, which contains SHUNT reproducibility by analytical method as measured by coefficient of variation (CV) and intraclass correlation coefficient (ICC) for patient groups (control, N=16; NASH, N=16; HCV, N=16). [Figure 8A] FIG. 1 shows eight graphs illustrating the correlation of DSI among second-generation cholate tests (DuO, TRIO, AI DuO, AI TRIO) and first-generation SHUNT test results analyzed by the MM and MMVd methods. [Figure 8B] FIG. 1 shows Table 10, which contains DSI reproducibility by analytical method as measured by coefficient of variation (CV) and intraclass correlation coefficient (ICC) for patient groups (control, N=16; NASH, N=16; HCV, N=16). [Figure 9A] FIG. 1 shows eight graphs illustrating the correlation of indexed hepatic reserve (HRindexed) among the results of second-generation cholate tests (DuO, TRIO, AI DuO, AI TRIO) and first-generation SHUNT cholate tests analyzed by the MM and MMVd methods. [Figure 9B] Figure 12 shows Table 12 with HR indexed reproducibility by analytical method as measured by coefficient of variation (CV) and intraclass correlation coefficient (ICC) for patient groups (control, N=16; NASH, N=16; HCV, N=16). [Figure 10A] Table 13A shows the reproducibility and diagnostic performance for predicting large esophageal varices. For the measurement of DSI, MMVd and TRIO (v1.0) cholate test version demonstrated similar ICC but improved CV compared to the MM method. For the measurement of SHUNT%, the diagnostic performance based on AUROC for MMVd and TRIO (v1.0) was comparable to MM in most cases and improved for MMVd. [Figure 10B]Figure 1 shows DSI values ​​measured by the DuO cholate test in Child-Pugh A cirrhosis subjects and control subjects. DSI from lean and overweight controls was plotted alongside subjects with no esophageal varices, small esophageal varices, or large esophageal varices (LEV). The DSI cutoff of 18.3 is shown as a horizontal line. A monotonic stepwise increase in DSI with increasing risk for LEV is seen. [Figure 10C] Figure 1 shows a graph of DSI values ​​measured by the DuO cholate test in Child-Pugh A cirrhosis subjects and control subjects against the probability of LEV based on DSI. The DSI cutoff of 18.3 is shown as a vertical line. DSI from DuO demonstrated a significant association with the endoscopic detection of LEV (p<0.001). [Figure 11] Figures 11A and 11B show pharmacokinetic clearance curves for various versions of the HepQuant liver function test for NASH subjects with F3 fibrosis. Figure 11A shows the pharmacokinetic clearance curve for the original cholate SHUNT liver function test (SHUNT V1.0), which measures the concentration of orally (PO) d4-CA and intravenously (IV) 13C-CA in the systemic circulation over 90 minutes. Figure 11B shows the pharmacokinetic clearance curve for the cholate SHUNT V1.1 liver function test, which uses patient physical characteristics to calculate initial 13C-CA concentrations, eliminating the need for 5-minute sample collection. Figure 11C shows the pharmacokinetic clearance curve for the cholate SHUNT V2.0 liver function test, which calculates oral and IV cholate clearance using samples collected at 20 and 60 minutes. Figure 11D shows the pharmacokinetic clearance curve for the cholate DuO liver function test, which calculates portal vein clearance from samples collected at 20 and 60 minutes and systemic clearance with induced IV. [Figure 12]

[0023] Figure 1 shows a schematic diagram of a compartmental model of dual cholate clearance describing the movement of cholate between the systemic, portal, and hepatic compartments. DIV = intravenous dose of C-cholate (SHUNT V2.0); DPO = oral dose of d4-CA (SHUNT V2.0 and DuO); qSP = splanchnic artery flow rate; qPS = portosystemic shunt flow rate; qPL = portal vein flow rate; qLS = hepatic return flow rate to the systemic circulation; qSL = hepatic artery flow rate; ClH = hepatic clearance. Reprinted with permission from Translational Research, Vol. 252, McRae, MP et al., Compartmental model describing the physiological basis for the HepQuant SHUNT test, pp. 53-63, Copyright (2023) with permission from Elsevier. [Figure 13] Figure 1 shows two graphs of fitting functions for the estimation of DSI (left panel) and portal vein HFR (right panel) using STAT. Scatter dots indicate values ​​for the PSC and REPRO studies (control, NASH, and HCV groups). [Figure 14] Figures 14A and 14B show the equivalence of the Next Generation Simplified Cholate Liver Function Test to SHUNT V1.1 for DSI by individual study and across all studies. Figure 14A shows plots of the two-tailed one-tailed t-test (TOST) analysis of SHUNT V2.0 for DSI. Figure 14B shows the bioequivalence analysis of SHUNT V2.0 for DSI. Figure 14C shows the TOST analysis of DuO for DSI. Figure 14D shows the bioequivalence analysis of DuO for DSI. Error bars represent 90% confidence intervals. Vertical dotted lines indicate the equivalence boundaries (TOST) or bioequivalence limits. REPRO = HepQuant Reproducibility Study; PSC = Primary Sclerosing Cholangitis Study; HALT-C = Hepatitis C Antiviral Long-Term Treatment Trial for Cirrhosis; ALL = All studies combined. [Figure 15]Figure 15 shows an equivalence analysis of SHUNT V2.0 versus SHUNT V1.1 for DSI. The Bland-Altman plot (Figure 15A) shows a graph of the method means versus the difference between methods including bias (solid black line), the 95% confidence interval for the mean difference (dotted line), and the equivalence bands (red line). The correlation plot (Figure 15B) shows the DSI values ​​from both methods and the Deming regression (solid line). Each point represents the value derived from a single test across all studies in the analysis. [Figure 16] Figure 16 shows an equivalence analysis of DuO vs. SHUNT V1.1 for DSI. The Bland-Altman plot (Figure 16A) shows the method means for the difference between methods including bias (solid black line), the 95% confidence interval for the mean difference (dotted line), and the equivalence bands (red line). The correlation plot (Figure 16B) shows the DSI values ​​from both methods and the Deming regression (solid line). Each point represents the value derived from a single test across all studies in the analysis. [Figure 17] Figure 17 shows an equivalence analysis of STAT vs. SHUNT V1.1 for DSI. The Bland-Altman plot (Figure 17A) shows the method means for the difference between methods including bias (solid black line), the 95% confidence interval for the mean difference (dotted line), and the equivalence bands (red line). The correlation plot (Figure 17B) shows the DSI values ​​from both methods and the Deming regression (solid line). Each point represents the value derived from a single test across all studies in the analysis. [Figure 18] Figure 18 shows an equivalence analysis of DuO vs. SHUNT V1.1 for hepatic reserve. The Bland-Altman plot (Figure 18A) shows the method means for the difference between methods including bias (solid black line), the 95% confidence interval for the mean difference (dotted line), and the equivalence bands (red line). The correlation plot (Figure 18B) shows the hepatic reserve values ​​from both methods and the Deming regression (solid line). Each point represents the value derived from a single test across all studies in the analysis. [Figure 19]Figures 19A and 19B show the equivalence of next-generation simplified liver function tests to SHUNT V1.1 for hepatic reserve (HR) by individual study and across all studies. Figure 19A shows a two-tailed one-tailed t-test (TOST) analysis of SHUNT 2.0 for HR. Figure 19B shows a bioequivalence analysis of SHUNT 2.0 for HR. Figure 19C shows a TOST analysis of DuO for HR. Figure 19D shows a bioequivalence analysis of DuO for HR. Error bars represent 90% confidence intervals. The dotted red lines indicate the equivalence boundaries (TOST) or bioequivalence limits. REPRO = HepQuant Reproducibility Study; PSC = Primary Sclerosing Cholangitis Study; HALT-C = Hepatitis C Antiviral Long-Term Treatment Trial for Cirrhosis; ALL = all studies combined. [Figure 20] Figure 20 shows an equivalence analysis of SHUNT V1.1 vs. SHUNT V1.0 for DSI. The Bland-Altman plot (Figure 20A) shows the method means for the difference between methods including bias (solid black line), the 95% confidence interval for the mean difference (dotted line), and the equivalence bands (red line). The correlation plot (Figure 20B) shows the DSI values ​​from both methods and the Deming regression (solid line). Each point represents the value derived from a single test across all studies in the analysis. [Figure 21A] FIG. 1 shows a graph of PSC progressor groups slow, intermediate, and rapid progressors as age against DSI values ​​from the DuO cholate test. [Figure 21B] FIG. 1 shows a graph of SHUNT% values ​​from the DuO cholate test versus PSC progressor groups slow, intermediate, and rapid progressors as age. [Figure 22A] FIG. 1 shows a graph of SHUNT% against probability of portal hypertension for PSC study subjects. [Figure 22B] FIG. 1 shows a graph of SHUNT% against probability of varicose veins for PSC study subjects. [Figure 23] FIG. 1 shows the timeline of the open-label active resmetirom treatment group in patients with well-compensated (Child-Pugh A [CP-A]) NASH cirrhosis. [Figure 24A]FIG. 1 shows pharmacokinetic clearance curves for the cholate SHUNT V2.0 liver function test, calculating oral and IV cholate clearance using samples collected at 20 and 60 minutes. [Figure 24B] Pharmacokinetic clearance curves for the cholate DuO liver function test are shown, calculating portal vein clearance from samples taken at 20 and 60 minutes and systemic clearance via induced IV. [Figure 25A]

[0023] Figure 1 shows the risk ACE calculated using the DuO cholate test after 28 weeks of resmetirom compared to baseline. The risk ACE from DuO was reduced by resmetirom treatment in 21 of 23 subjects. [Figure 25B]

[0023] Figure 1 shows risk ACE calculated using DuO cholate test after 48 weeks of resmetirom compared to baseline. At 48 weeks, risk ACE decreased in 19 of 23 subjects. [Figure 26A]

[0023] Figure 1 shows the risk ACE calculated using the SHUNT V2.0 cholate study after 28 weeks of resmetirom compared to baseline. The risk ACE from SHUNT V2.0 decreased in 20 of 23 subjects at 28 weeks. [Figure 26B]

[0023] Figure 1 shows the risk ACE calculated using the SHUNT V2.0 cholate study after 48 weeks of resmetirom compared to baseline. The risk ACE from SHUNT V2.0 decreased in 19 of 23 subjects at 48 weeks. DETAILED DESCRIPTION OF THE INVENTION

[0063] Detailed Description of the Invention Current noninvasive liver tests are surrogates for fibrosis and do not measure function. The HepQuant platform of noninvasive cholate liver function testing uniquely assesses both liver function and physiology through hepatic uptake of stable isotopes of cholate. However, the prototype HepQuant SHUNT test (SHUNT V1.0), described in U.S. Patent Nos. 8,613,904 and 8,778,299, is cumbersome to administer, requiring intravenous and oral administration of cholate and peripheral venous blood samples at six time points over a 90-minute period.

[0064] To reduce the burden of administering the SHUNT test, we investigated whether the oral-only (DuO) cholate liver function test and other simplified versions of the test could provide a reproducible measurement of liver function while maintaining comparability of results. The simplified test versions include SHUNT V1.1 (oral and IV dosing, but four blood samples), SHUNT V2.0 (oral and IV dosing, but only two blood samples over 60 minutes), and DuO (oral dosing and two blood samples over 60 minutes).

[0065] The simplified test methods, particularly SHUNT V2.0 and DuO, are easier to perform and less invasive, and therefore have the potential to be more widely accepted by the healthcare providers who administer the tests and by the patients who undergo the tests. The HepQuant test versions are presented in Table 1A, which outlines the analytical methods for the number and route of administration of cholate isotope doses, the number of blood samples, and the duration of the test time in minutes.

[0066] [Table 1]

[0067] The first-generation cholate SHUNT liver function test (SHUNT V1.0) simultaneously measures portal and systemic clearance and involves an intravenous (IV) dose of carbon-13 labeled cholate (13C-CA), a concurrent oral dose of deuterium-labeled cholate (d4-CA), and six peripheral venous blood draws over 90 minutes. The SHUNT V1.0 test can be analyzed by the MM method.

[0068] Cholate SHUNT V1.0 (Figure 11A) liver function test and MM analysis methods were used for IV and oral area under the curve (AUC IV , AUC Oral (2007;26:401-10). Peripheral venous blood samples were obtained at 0, 5, 20, 45, 60, and 90 minutes within specified time windows, and the concentration of administered cholate was measured by liquid chromatography / mass spectrometry (LC / MS). After IV infusion, residual 13C-CA in the sampling catheter and blood collection beyond the time window, particularly for the 5-minute sample (±1 minute), were sources of error in a few studies, which may have compromised the results.

[0069] Cholate SHUNT V1.1 (Figure 11B) liver function test was performed using MM to exclude 5-minute samples from the calculation. VdThis is a cholate liver function test using analytical methods. The initial 13C-CA concentration at 0 min is calculated from the administered dose and blood volume estimation from body mass index (BMI). Lemmens et al., Estimating blood volume in obese and morbidly obese patients. Obes Surg. 2006;16:773-6. The 5-min 13C-CA concentration is then estimated by log-linear regression between the 0-min and 20-min concentrations, and the 5-min oral concentration is approximated as 15% of the 20-min d4-CA concentration. The AUC IV and AUC Oral is estimated by the same minimal model equation as in SHUNT V1.0.

[0070] Cholate SHUNT V2.0 (Figure 11C) (also known as TRIO V1.0) is a cholate liver function test that implements (1) a compartmental model of portal venous cholate clearance using hepatic flow and physiological assumptions to predict oral clearance curves and (2) a non-compartmental exponential fit for systemic cholate clearance. The compartmental model for measuring portal venous clearance (Figure 12) describes the flow between the systemic (S), portal (P), and hepatic (L) compartments represented by a system of first-order ordinary differential equations (Equations 1B, 2B, and 3), where q is the flow rate between the compartments, V is the volume of the compartment, C is the concentration of d4-CA in the compartment, and Cl H is the hepatic clearance and D PO,rate is the rate of orally administered d4-CA entering the portal compartment.

[0071]

number

number

number

[0072] The system of ordinary differential equations is solved numerically and the AUC Oral The d4-CA concentration in the whole body compartment is integrated over 180 min to calculate σ.

[0073] Noncompartmental analysis to measure systemic clearance involves exponential fitting of systemic cholate clearance using 20- and 60-minute 13C-CA concentrations. The exponential fitting divides the curve into three clearance phases: rapid (Y0), moderate (Y1), and slow (Y2). Equations 4C, 5B, and 6B define the systemic concentration of 13C-CA over time.

[0074]

number

number

number

[0075] where t is time (0-20 min, 20-60 min, and 60-180 min for Y0, Y1, and Y2, respectively), C0 is the initial concentration of 13C-CA, and C 20 and C 60 are the measured 20-minute and 60-minute concentrations of 13C-CA, and T 20 and T 60 are the actual collection times of the 20-minute and 60-minute blood samples. IV The systemic concentration of 13C-CA is integrated to calculate ∑ ∑ C-CA = ...

[0076] The DuO test (FIG. 11D) (also known as DuO V1.0 or the dual-sample oral cholate tolerance test) is an oral-only cholate liver function test that involves the administration of a single oral dose (d4-CA at 0 min) and the collection of two blood samples (e.g., at 20 and 60 min) to quantify portal venous HFR and estimate systemic HFR. These analyses involve the same compartmental model of portal venous cholate clearance as SHUNT V2.0. Systemic clearance is calculated by first estimating derived IV concentrations at 20 and 60 min using a linear regression model (Table 14) that includes body weight, BMI, the actual time of the 20-min blood sample, and d4-CA concentrations at 20 and 60 min (see Example 2 for a detailed description of the linear model and regression coefficients). The derived IV concentrations are then used in the same noncompartmental analysis as SHUNT V2.0.

[0077] The STAT V1.0 test (also known as the cholate STAT test or STAT) is an oral-only cholate liver function test that involves the administration of a single oral dose (e.g., d4-CA at 0 minutes) and the collection of a single blood or serum sample (e.g., at 60 minutes). The STAT test is simply the d4-CA concentration at 60 minutes normalized to 75 kg body weight by the calculation ([d4-CA] x kg body weight / 75 kg). Values ​​derived from the STAT test can be used by themselves or in the estimation of portal vein HFR and DSI (Figure 13).

[0078] Liver function test parameters

[0079] The following parameters were calculated by the SHUNT and DuO studies and have previously been demonstrated to be related to liver function:

[0080] Portal hepatic filtration rate (HFR) P ) is the portal venous clearance (in mL min ) adjusted for body weight calculated by Equation 7B. -1 kg -1 ) and D PO is the oral dose.

[0081]

number

[0082] Whole body hepatic filtration rate (HFR) S ) is the measured systemic clearance (in mL min ) adjusted for body weight as calculated by Equation 8B. -1 kg -1 ) and D IV is the intravenous dose.

[0083]

number

[0084] SHUNT% is the estimated absolute bioavailability of orally administered d4-CA or equivalently the ratio of systemic and portal venous HFR (Equation 9B).

[0085]

number

[0086] The DSI is a score that indicates overall liver function, including both portal and systemic HFR. The DSI conveys unique information about the fibrosis stage and clinical stage of cirrhosis, and modeling these outputs for the prediction of clinical outcomes generated the DSI in Equation 10B [8, 10-12]. Here, HFR P,max and HFR S,max is the upper limit of clearance relative to healthy controls, and A is a coefficient for scaling the DSI from 0 to 50.

[0087]

number

[0088] Hepatic reserve is a numerical index of overall liver health with values ​​from 0 to 100 (Equation 11B). Here, hepatic reserve is the HFR indexed to lean control minus 1 standard deviation by a scaling factor A to scale hepatic reserve from 100 to 0. P and HFR S (HFR P,lean and HFR S,lean ) is used. P,lean Any HFR exceeding P The value is HFR P,lean Similarly, the HFR S,lean Any HFR exceeding S The value is HFR S,lean Therefore, any patient with both HFR values ​​above these limits would be calculated to have an HR of 100.

[0089]

number

[0090] RCA-20 is the residual 13C-CA at 20 minutes as a percentage of the initial estimated 13C-CA immediately after IV administration.

[0091] Risk-ACE (clinical events per person-year) is derived from a Poisson regression model as a function of DSI.

[0092] STAT can be derived from SHUNT, DuO, or STAT tests as d4-CA concentration at 60 minutes normalized to 75 kg body weight.

[0093] The reproducibility of the test parameters DSI, SHUNT%, portal HFR, systemic HFR, hepatic functional reserve, RCA-20, risk ACE, STAT, STAT-estimated DSI, and STAT-estimated portal HFR as calculated using the SHUNT V1.0, SHUNT V1.1, SHUNT V2.0, and DuO analysis methods provided herein is shown in Example 2.

[0094] The present disclosure provides a series of second-generation liver function tests that correlate with first-generation liver function tests (cholate STAT, FLOW, and SHUNT tests) but provide accurate measures of liver function that are significantly simplified in terms of test administration.

[0095] Liver function test parameters (DSI, HFR, SHUNT, HR, etc.) were measured by oral and IV discriminative cholate clearance area under the curve (AUC Oral , AUC IV ) requires accurate and reliable measurement.

[0096] The present disclosure provides simplified Duo and TRIO liver function tests that require only a limited number of data points (only two oral data points for Duo, two oral + one IV data point for TRIO), and specialized analytical techniques for fitting systemic and portal clearance curves therefrom. Analytical methodologies are provided herein for each test.

[0097] Briefly, the methods provided herein involve the use of cholate DuO liver function tests, cholate TRIO liver function tests, cholate AI liver function tests, and cholate Vd-based minimal model (MM). Vd ) including liver function tests.

[0098] The cholate DuO liver function test (also known as the dual-sample oral cholate tolerance test, "DuO," "DUO," "DuO test," "DuO cholate test," "HepQuant DUO," or "HepQuant DuO") is a compartmental model of portal venous cholate clearance that uses hepatic flow and physiological assumptions to predict oral clearance curves, involving measuring only two orally administered distinct cholate (e.g., d4-CA) concentration time points (e.g., 20 and 60 minutes) (i.e., DuO liver function test v1.0). The DuO v1.0 test involves the administration of a single oral dose of distinct cholate (e.g., d4-CA at 0 minutes) and the collection of two blood draws at the first and second time points (e.g., 20 and 60 minutes). The DuO v2.0 test involves the administration of two oral doses of a first and a second distinguishable cholate at a first and a second time point, respectively (e.g., d4-CA at 0 minutes and 13C-CA at 40 minutes), and the collection of a single blood draw at a single time point (e.g., 60 minutes). The DuO test quantifies portal vein HFR and estimates systemic HFR, DSI, SHUNT%, and hepatic reserve (HR) using only oral doses. The DuO dual oral cholate clearance test does not require measurement of intravenously administered distinguishable cholate.

[0099] The Cholate SHUNT V2.0 Liver Function Test (also known as the "TRIO V1.0 Test," "Cholate TRIO V1.0 Liver Function Test," and "HepQuant TRIO") is a non-compartmental method involving exponential fitting of systemic cholate clearance (i.e., the TRIO Liver Function Test v1.0), which involves measuring a single (e.g., 20-minute) intravenous dose of a distinguishable cholate (e.g., 13C-CA) concentration time point. The SHUNT V2.0 Test (TRIO V1.0 Test) involves the administration of a single oral dose of a first distinguishable cholate (e.g., d4-CA at 0 minutes) at a first time point and a single intravenous dose of a second distinguishable cholate (e.g., 13C-CA at 0 minutes) at a second time point, and the collection of two blood samples (e.g., at 20 minutes and 60 minutes).

[0100] The Cholate SHUNT V3.0 Liver Function Test (also known as the TRIO V2.0 Test or the Cholate TRIO V2.0 Liver Function Test) involves administering two oral doses of two different identifiable cholate salts (e.g., d4-CA at 0 minutes, d2-CA at 40 minutes, or d5-CA at 40 minutes) at time points one and two, and one intravenous dose of a third identifiable cholate salt (e.g., 13C-CA at 40 minutes) at time point three, and collecting one blood sample (e.g., at 60 minutes). The second and third time points can be simultaneous or substantially simultaneous. The TRIO Test quantifies portal vein HFR, systemic HFR, DSI, SHUNT%, and hepatic reserve (HR) using the orally and intravenously administered cholate doses.

[0101] The cholate AI liver function test involves a function-fitting neural network trained to predict response curves as calculated by the minimal model approach (i.e., DuO v1.0 and TRIO V1.0 cholate tests) when provided with a limited set of inputs.

[0102] Cholate SHUNT Vd-based minimal model (MM Vd ) SHUNT V1.1, also known as the Liver Function Test, is an adaptation of the previously validated minimal model method that uses the volume of distribution (Vd) estimated from body mass index (BMI) to inform the initial 13C-CA concentration at 0 minutes.

[0103] definition As used herein, "a" or "an" may mean one or more than one of an item.

[0104] The term "about," when referring to any numerical parameter, means ±10% of the numerical value. For example, the phrase "about 60 minutes" means 60 minutes ±6 minutes.

[0105] All patents, patent applications and publications referenced herein are hereby incorporated by reference in their entirety.

[0106] The term "accuracy" (measurement) as used herein means the closeness of agreement between the value of a measured quantity and the value of the true quantity of the measurement.

[0107] The term "acceptability" as used herein is based on specific criteria that establish minimum operating characteristics for a measurement procedure.

[0108] The term "match rate" as used herein means the closeness of agreement between individual test / measurement results obtained under specified conditions.

[0109] The term "accuracy," as used herein, means the closeness of agreement between the expected and true values ​​of a test or measurement result.

[0110] The term "measurement" is used when referring to the quantity intended to be measured instead of the analyte (the component nominally represented by a measurable quantity).

[0111] The term "verification," as used herein, focuses on whether the details of a measurement procedure can be accomplished, while the term "validation" confirms that the procedure is fit for its intended purpose.

[0112] The term "measurement procedure" means a detailed description of a measurement according to a given measurement method based on one or more measurement principles and models and including any calculations to obtain a measurement result.

[0113] As used herein, "clearance" can mean the removal of a substance from one location to another.

[0114] As used herein, the term "simultaneously," when referring to two or more events, means occurring within 10 minutes or less, within 5 minutes, or within about 3 minutes of each other.

[0115] As used herein, the terms "patient" or "subject" include, but are not limited to, humans, and may also include other mammals, or pet or exotic animals, such as dogs, cats, ferrets, rabbits, pigs, horses, cows, birds, or reptiles.

[0116] The acronym "HALT-C" stands for the Hepatitis C Antiviral Long-Term Treatment Trial for Cirrhosis. The HALT-C trial was a large, prospective, randomized, controlled trial of long-term, low-dose pegylated interferon therapy in patients with advanced hepatitis C who had not had a sustained virological response to a previous course of interferon-based therapy. The NIH-funded Hepatitis C Antiviral Long-Term Treatment for Cirrhosis (HALT-C) trial investigated whether long-term use of antiviral therapy (maintenance treatment) slows the progression of liver disease. In non-cirrhotic patients with significant fibrosis, effective maintenance treatment was expected to slow or halt histological progression to cirrhosis as assessed by serial liver biopsies. However, tracking disease progression using biopsies carries the risk of complications and possible mortality. In addition, sampling error and variability in pathological interpretation of liver biopsies limit the accuracy of histological assessments and endpoints. Histological endpoints are unreliable because advanced fibrosis is already present and cannot detect changes in fibrosis related to treatment or disease progression. Therefore, the standard endpoint for effective response to maintenance therapy in cirrhotic patients is the prevention of clinical decompensation (ascites, variceal bleeding, and encephalopathy) and stabilization of liver function as clinically measured by the Childs-Turcotte-Pugh (CTP) score. However, clinical endpoints and CTP scores have been known to be insensitive parameters of disease progression. Dual-isotope technology utilizing distinguishable cholate was used in the development of the SHUNT test and in conjunction with the HALT-C trial. The term "SHUNT test" refers to the previously disclosed QLFT (quantitative liver function test), which is used as a comprehensive assessment of hepatic blood flow and liver function. The SHUNT test is used to determine the clearance of orally and intravenously administered distinguishable cholate in subjects with and without chronic liver disease. The SHUNT fraction or percent quantifies the extravasation of PO d4-cholate into the systemic circulation as the ratio of the clearance of intravenously administered 13C-cholate to the clearance of orally administered d4-cholate.In the SHUNT test, at least five blood samples are collected from a patient at intervals of at least about 90 minutes after oral and intravenous administration of a discriminative cholate. The SHUNT test is disclosed in U.S. Pat. No. 8,613,904 to Everson et al., which is incorporated herein by reference. These studies demonstrated reduced clearance of cholate in patients with either hepatocellular injury or portosystemic shunting. "SHUNT test value" refers to a number (in %). The term "SHUNT%" represents a quantitative measurement of portosystemic shunting. SHUNT% is a measure of the percentage of extravasation of orally administered d4-cholate. The first-pass hepatic elimination of cholate in percent of orally administered cholate is defined as (100%-SHUNT). The SHUNT test method is disclosed in U.S. Patent Nos. 8,613,904, 9,639,665, 8,778,299, 9,417,230, and 10,215,746, each of which is incorporated herein by reference in its entirety. Analysis of the sample for stable isotope-labeled cholate is performed, for example, by GC-MS after sample derivatization, or by LC-MS without sample derivatization, or by LC-MS / MS or MS / MS as disclosed herein. The ratio of the AUC of orally administered cholate to intravenously administered cholate, corrected for the administered dose, defines the cholate shunt. The cholate shunt is determined by the formula AUC = AUC + 1 / (A ... oral / AUC iv ×Dose iv / Dose oral × 100%, where AUC oral is the area under the curve of the serum concentration of orally administered cholic acid, and AUC iv is the area under the curve for intravenously administered cholic acid.

[0117] The SHUNT test allows for the measurement of first-pass hepatic elimination of bile acids from the portal circulation. The flow-dependent first-pass elimination of bile acids by the liver ranges from approximately 60% for unconjugated dihydroxy bile acids to approximately 95% for glycine-conjugated cholate. Free cholate used herein has a reported first-pass elimination of approximately 80%, which closely matches the previously observed first-pass elimination of approximately 83% in healthy controls. After uptake by the liver, cholic acid is efficiently conjugated to either glycine or taurine and secreted into bile. Physicochemically, cholic acid can be easily separated from other bile acids and bile acid or cholate conjugates using chromatographic methods.

[0118] The term "cholate disappearance rate" k elim minutes -1 represents the first phase of elimination of intravenously administered 13C-cholate, calculated from the linear regression of [13C-cholate] against time (using only the 5-minute and 20-minute time points). Intravenously administered 13C-cholate is rapidly delivered to the liver via the hepatic artery. In contrast, the same 13C-cholate passes slowly to the liver via the portal vein due to the capacitance of the splanchnic vascular bed. Therefore, the first phase of cholate elimination is more dependent on clearance from the hepatic artery than from the portal vein.

[0119] The term "distribution volume" V d , (L kg -1 ) represents the volume of the body into which cholate is distributed.

[0120] The acronym "IV" or "iv" refers to the intravenous route of administration.

[0121] The acronym "PO" means oral route of administration.

[0122] The acronym "PHM" stands for perfused hepatic mass.

[0123] The acronym "SF" refers to the shunt fraction as in, for example, liver SF or cholate SF.

[0124] The acronym "ROC" stands for receiver operating characteristic. An ROC curve is a graphical plot illustrating the performance of a binary classifier system when the discrimination threshold is varied. An ROC curve is constructed by plotting the proportion of true positives among positives (TPR = true positive rate) against the proportion of false positives among negatives (FPR = false positive rate) at various threshold settings. Sensitivity is the probability of a positive test result, or a value above the threshold, among disease-containing cases. Sensitivity is defined as the true positive rate (TPR): TPR = TP / P = TP / (TP + FN). The false positive rate (FPR) is FPR = FP / N = FP / (FP + FN). Accuracy (ACC) is defined as ACC = (TP + TN) / (P + N). Specificity is the probability of a negative test result, or a value below the threshold, among disease-free cases. Specificity (SPC), or true negative rate (TN), is defined as SPC = TN / N = TN / (FP + TN) = 1 - FPR. Positive predictive value (PPV) is defined as PPV = TP / (TP + FP). Negative predictive value (NPV) is defined as NPV = TN / (TN + FN).

[0125] The c-statistic is the area under the ROC curve, or "AUROC" (area under the receiver operating characteristic curve), which ranges from 0.5 (no discrimination) to a theoretical maximum of 1 (perfect discrimination).

[0126] The terms "treating" or "treatment" of a condition or disease includes: (i) preventing the condition or disease, i.e., preventing the clinical symptoms of the condition or disease from developing in a subject who may be exposed to or susceptible to the condition or disease, but who has not yet experienced or displayed symptoms of the condition or disease; (ii) inhibiting the condition or disease, i.e., arresting the development of the condition or disease, or its clinical symptoms; or (iii) alleviating the condition or disease, i.e., causing the temporary or permanent regression of the condition or disease, or its clinical symptoms.

[0127] The term "sustained virologic response" (SVR) is used to describe a desired response in a patient, for example, when the hepatitis C virus is undetectable in the blood six months after completing treatment. Conventional treatment with interferon and ribavirin does not necessarily eliminate or eliminate the hepatitis C virus. Sustained virologic response is associated with a very low incidence of relapse. SVR is used to evaluate new medical treatments and compare them with approved therapies.

[0128] The term "distinguishable cholate" or "distinguishable cholate compound" can be any cholate compound that is analytically distinguishable from naturally occurring cholate in a subject's blood or serum. A distinguishable cholate compound can be a labeled cholate compound or an unlabeled cholate compound. A distinguishable cholate compound can be a fluorescent moiety-labeled cholate compound. Various fluorescent probes, such as fluorescein, Alexa Fluor dyes, quantum dots, etc., are commercially available. A distinguishable cholate is an isotopically labeled cholate compound. A distinguishable cholate compound can be a cholate compound labeled with a stable isotope (e.g., 13 C. 2 H, 18 O) or radioisotopes (e.g., 14 C. 3 H). Differentiable cholate compounds are commercially available and can be purchased (e.g., CDN Isotopes Inc., Quebec, CA).

[0129] The distinguishable cholate compound can be a stable isotope-labeled cholate compound. The distinguishable cholate can be selected from any known safe, non-radioactive stable isotope of cholic acid. In one specific embodiment, the distinguishable cholate compound is 2,2,4,4-cholic acid-2,2,4,4-d4 (D4-CA), also known as cholic acid-2,2,4,4-d4 (D4-CA). 2 In another specific embodiment, the distinguishable cholate compound is cholic acid-24- 13 C( 13 24-, also known as C-CA 13In another specific embodiment, the distinguishable compound is 2,2,3,4,4-C cholic acid, also known as cholic acid-2,2,3,4,4-d5 (D5-CA). 2 H cholic acid.

[0130] In some embodiments, the distinguishable cholate compound can be selected from any of the following labeled compounds: cholic acid, any glycine conjugate of cholic acid, any taurine conjugate of cholic acid; chenodeoxycholic acid, any glycine conjugate of chenodeoxycholic acid, any taurine conjugate of chenodeoxycholic acid; deoxycholic acid, any glycine conjugate of deoxycholic acid, any taurine conjugate of deoxycholic acid; or lithocholic acid, or any glycine or taurine conjugate thereof. The distinguishable cholate compound can be selected from those described in WO 2021 / 207683 A1, HepQuant, LLC, Everson and Helmke, which is incorporated herein by reference in its entirety.

[0131] Cholate occurs naturally and is not known to have any adverse or harmful effects when given intravenously or orally in the doses used in the inventive or comparative studies herein. The serum cholate concentrations achieved by either intravenous or oral doses are similar to the serum concentrations of bile acids that occur after ingestion of a fatty meal. Because cholate occurs naturally with a pool size in humans of 1-5 g, the 20 and 40 mg doses of labeled cholate used herein are unlikely to be harmful.

[0132] The term "oral cholate clearance" (Cl oral"Oral cholate clearance" refers to the clearance of an orally administered cholate compound from a subject's body as measured by blood or serum samples from the subject. Oral cholate clearance is used as a measure of portal blood flow. Orally administered cholic acid is absorbed through the epithelial lining cells of the small intestine, binds to albumin in the portal blood, and is transported to the liver via the portal vein. Approximately 80% of cholic acid is extracted from the portal blood on its first pass through the liver. Cholic acid that escapes hepatic extraction resides in the liver via the hepatic vein and flows into the vena cava, returning to the heart for delivery to the systemic circulation. The area under the curve (AUC) of peripheral venous concentration versus time after oral administration of cholic acid quantifies the proportion of cholic acid that escapes hepatic extraction and defines "oral cholate clearance."

[0133] The terms "portal hepatic filtration rate," "portal HFR," and "FLOW test" (HFRp) refer to oral cholate clearance (portal hepatic filtration rate; portal HFR) used as a measure of portal blood flow, or portal circulation, obtained from analysis of the concentration of an identifiable cholate compound, e.g., an identifiable cholate, in at least five blood samples taken from a subject over a period of, e.g., about 90 minutes, following oral administration of the identifiable cholate compound. The units of portal HFR values ​​are typically expressed as mL / min / kg, where kg refers to the subject's kg body weight. "Portal HFR" mL min -1 kg -1 can be used to model the independent apparent clearance of orally administered d4-cholate, adjusted for body weight and calculated from dose / AUC. The FLOW test method is disclosed in U.S. Patent Nos. 8,778,299, 9,417,230, and 10,215,746, each of which is incorporated by reference in its entirety.

[0134] Term “Whole body HFR” (HFR) mL min -1 kg -1 can be used to model the independent clearance of intravenously infused 13C-cholate, adjusted for body weight and calculated from dose / AUC. "Systemic HFR" mL min-1 kg -1 can be used to model the independent clearance of intravenously infused 13C-cholate, adjusted for body weight and calculated as dose / AUC.

[0135] The term "STAT test" (STAT) refers to an estimate of portal vein blood flow by analysis of a single patient blood sample taken at a specified time after oral administration of a differentiable cholate. In one aspect, a STAT test refers to the analysis of a single blood sample taken at a specific time point after oral administration of a differentiable cholate. In one specific aspect, the STAT test is a simplified and convenient test intended for screening purposes that can reasonably estimate portal vein blood flow (estimated flow rate) from a single blood sample obtained 60 minutes after oral administration of deuterated cholate. In some embodiments, the STAT is the d4-cholate concentration in the 60-minute blood sample. The STAT correlates well with and can be used to estimate DSI. STAT test values ​​are typically expressed as concentrations, e.g., micromolar (μM) concentrations. STAT test methods are disclosed in U.S. Pat. Nos. 8,961,925 and 10,222,366, each of which is incorporated herein by reference in its entirety. STAT test values ​​can be used to estimate portal vein HFR as provided in U.S. Patent Nos. 8,961,925 and 10,222,366. STAT test values ​​in a patient can be used to estimate DSI values ​​in a patient as provided herein.

[0136] The term "DSI test" (DSI) refers to a disease severity index test derived from one or more liver function test results based on hepatic blood flow. The DSI score is a function of the sum of cholate clearance from the systemic and portal circulations adjusted for disease severity ranging from healthy subjects to end-stage liver disease. The DSI is a unit-free score that represents a quantitative measurement of liver function. A disease severity index (DSI) value can be obtained from a patient with or at risk of chronic liver disease by a method comprising: (a) obtaining one or more liver function test values ​​from a patient with chronic liver disease, wherein the one or more liver function test values ​​are obtained from one or more liver function tests selected from the group consisting of SHUNT, portal vein hepatic filtration rate (portal HFR), and total body hepatic filtration rate (total body HFR); and (b) using a disease severity index equation (DSI equation) to obtain a DSI value from the patient, wherein the DSI equation comprises one or more terms and constants for obtaining a DSI value, and at least one term of the DSI equation independently represents the liver function test value from the patient or the mathematically transformed liver function test value from step; and at least one term of the DSI equation is multiplied by a coefficient specific to the liver function test.DSI is an index or score that includes cholate clearance from both systemic and portal circulation. DSI ranges from 0 (healthy) to 50 (severe end-stage disease) and is calculated from both HFRs. Based on the reproducibility of DSI values, the minimum detectable difference indicating a change in liver function in a subject may be about 1.5 points, about 2 points, or about 3 points. DSI test methods and equations are disclosed in U.S. Patent Nos. 9,091,701, 9,759,731, and 10,520,517, each of which is incorporated herein by reference in its entirety. A method for estimating a patient's DSI value from STAT test values ​​is also provided herein.

[0137] The term "hepatic reserve" refers to the percentage of maximum liver function capacity measured by DSI, and the indexed hepatic reserve can be normalized to the DSI range in lean subjects. HR (algebraic) is simply the algebraic transformation of the DSI value in a subject: HR = [100 - (2 x DSI)]. The indexed HR is normalized to the results in a cohort of normal lean controls.

[0138] The term "RCA20" refers to the amount of an intravenously administered identifiable compound, eg, an identifiable cholate compound, eg, 13C-CA, that remains in the circulation 20 minutes after intravenous infusion.

[0139] The term "quantitative liver function test" (QLFT) refers to an assay that measures the liver's ability to metabolize or extract a test compound, can identify patients with impaired liver function at an earlier stage of disease, and may define the risk for cirrhosis, splenomegaly, and varices. One of these assays is the cholate shunt assay, in which cholate clearance is assessed by analyzing a body fluid sample after exogenous cholate is taken up by the body.

[0140] The term "Ishak fibrosis score" is used in reference to a scoring system that measures the degree of liver fibrosis (scarring) caused by chronic necroinflammation. A score of 0 represents no fibrosis, and 6 is established fibrosis. Scores of 1 and 2 indicate mild portal fibrosis; stages 3 and 4 indicate moderate (bridging) fibrosis. A score of 5 indicates nodular and incomplete cirrhosis, and 6 is established cirrhosis.

[0141] The term "Childs-Turcotte-Pugh (CTP) score" or "Child-Pugh score" refers to a classification system used to assess the prognosis of chronic liver disease, as provided by Pugh et al., Transection of the esophagus for bleeding esophageal varices. Br J Surg 1973;60:646-649, incorporated herein by reference. The CTP score includes five clinical measures of liver disease; each measure is scored from 1 to 3, with 3 being the most severe. The five scores are added to determine the CTP score. The five clinical measures include total bilirubin, serum albumin, prothrombin time international normalized ratio (PT INR), ascites, and hepatic encephalopathy. The CTP score is a scoring system used to stratify the severity of end-stage liver disease. Chronic liver disease is classified into Child-Pugh classes A to C, which utilize additional scores. Child-Pugh class A means a CTP score of 5 to 6. Child-Pugh class B means a CTP score of 7 to 9. Child-Pugh class C means a CTP score of 10 to 15. A website calculates the risk of postoperative mortality in patients with cirrhosis: http: / / mayoclinic.org / meld / mayomodel9.html

[0142] The term "Model for End-Stage Liver Disease" (MELD) refers to a scoring system used to assess the severity of chronic liver disease. MELD was developed to predict death within three months of surgery in patients undergoing transjugular intrahepatic portosystemic shunting (TIPS) for liver transplantation. MELD is also used to determine prognosis and priority for receiving a liver transplant. MELD uses patient values ​​for serum bilirubin, serum creatinine, and prothrombin time international normalized ratio (INR) to predict survival. The scoring system is used by the United Network for Organ Sharing (UNOS) and Eurotransplant to prioritize liver transplant allocation, replacing the older Child-Pugh score. See UNOS (2009-01-28) "MELD / PELD calculator documentation," incorporated herein by reference. For example, in interpreting the MELD score in hospitalized patients, the three-month mortality rate is 71.3% for a MELD score of 40 or greater.

[0143] The term "standard sample" refers to a sample containing a known concentration of an analyte that is used for comparison purposes when analyzing a sample containing an unknown concentration of the analyte.

[0144] The term "chronic hepatitis C" (CHC) refers to a chronic liver disease caused by a viral infection that leads to liver inflammation, damage to the liver, and cirrhosis. Hepatitis C is an infection caused by a blood-borne virus that attacks the liver and causes inflammation. Many people infected with hepatitis C virus (HCV) do not show symptoms until liver damage appears during a routine medical checkup, sometimes years later.

[0145] The term "fatty liver disease" (SLD) encompasses various etiologies of hepatic steatosis.

[0146] The term "alcoholic steatohepatitis" (ASH) refers to a chronic inflammatory condition of the liver caused by excessive alcohol consumption. Progressive inflammatory liver damage is associated with long-term heavy ethanol intake and can progress to cirrhosis.

[0147] The term "metabolic dysfunction-associated steatohepatitis" (MASH), formerly known as "nonalcoholic steatohepatitis" (NASH), refers to a severe, chronic condition of liver inflammation that progresses from a less severe, simple fatty liver condition called steatosis. Simple steatosis (alcoholic fatty liver) is an early, reversible consequence of excessive alcohol consumption. In people who do not drink large amounts of alcohol, the cause of fatty liver disease is less clear but may be related to factors such as obesity, hyperglycemia, insulin resistance, or high levels of blood triglycerides. In certain cases, fat accumulation may be accompanied by inflammation and scarring in the liver. This more severe form of the disease is called metabolic dysfunction-associated steatohepatitis (MASH), formerly known as nonalcoholic steatohepatitis (NASH). MASH is associated with a much higher risk of liver fibrosis and cirrhosis than MASLD. Patients with MASH have an elevated risk of hepatocellular carcinoma. MASLD can progress to MASH, which is associated with fibrosis, cirrhosis, and hepatocellular carcinoma.

[0148] The term "metabolic dysfunction-associated fatty liver disease" (MASLD), formerly known as "nonalcoholic fatty liver disease" (NAFLD), refers to a common chronic liver disease characterized in part by a fatty liver condition with associated risk factors of obesity, metabolic syndrome, and insulin resistance. Both MASLD and MASH are often associated with obesity, diabetes, and asymptomatic elevations of serum ALT and gamma-GT. Ultrasound monitoring can suggest the presence of fatty infiltration of the liver; differentiation between MASLD and MASH typically requires a liver biopsy.

[0149] The term "metabolic dysfunction-associated alcoholic liver disease" (Met-ALD) refers to MASLD patients who consume higher amounts of alcohol per week (more than 140 g / week for women and more than 210 g / week for men).

[0150] The term "primary sclerosing cholangitis" (PSC) refers to a chronic liver disease caused by progressive inflammation and scarring of the bile ducts in the liver. Scarring of the bile ducts can block bile flow, causing cholestasis. Inflammation can lead to cirrhosis, liver failure, and liver cancer. Chronic bile duct obstruction leads to portal tract fibrosis and ultimately biliary cirrhosis and liver failure. The definitive treatment is liver transplantation. Indications for transplantation include recurrent bacterial cholangitis, recurrent jaundice after medical and endoscopic treatment, decompensated cirrhosis, and complications of portal venous hypertension (PHTN). PSC progresses to variceal ascites and encephalopathy through chronic inflammation, fibrosis / cirrhosis, alterations in portal circulation, portal hypertension, and portosystemic shunting. Alterations in portal flow are a sign of clinical complications.

[0151] Any suitable analytical method known in the art can be used to quantify distinguishable cholate compounds in blood or serum samples. For example, detection and quantification of distinguishable cholate compounds in a sample can include high-performance liquid chromatography (HPLC), HPLC-diode array detection (HPLC-DAD), HPLC-fluorescence, ultra-performance liquid chromatography (UPLC), mass spectrometry (MS), GC-MS, LC-MS, LC-MS / MS, surface-enhanced Raman scattering (SERS), immunoassays using, for example, isolated antibodies, monoclonal antibodies, or antigen-binding fragments thereof, single-domain antibodies, aptamers, etc. Methods for detection and quantification of distinguishable cholate compounds are described, for example, in US20210318274, which is incorporated herein by reference in its entirety.

[0152] Blood or serum samples for use in the methods of the present invention can be collected from a subject by any method known in the art. See, for example, WHO guidelines on drawing blood: best practices in phlebotomy, World Health Organization, 2010, Geneva, Switzerland, or BP-EIA: Collecting, processing, and handling venous, capillary, and blood spot samples, PATH, 2005. For example, venipuncture using a needle and syringe or an indwelling catheter, arterial blood collection, pediatric or neonatal blood collection, or capillary collection can be used. The choice of site and procedure can depend on the volume of blood required for the procedure and clinical test to be performed. For example, a venous site, capillary collection, or fingerstick or heelstick, also known as skin puncture, can be used.

[0153] Whole blood samples can be obtained by venipuncture, collected in anticoagulant-containing vacutainer tubes, and refrigerated during storage and transport. Blood samples can be further processed into different fractions. The blood or serum sample can be a peripheral blood sample. The sample can be a transcutaneous blood sample. Various commercially available devices are available for obtaining transcutaneous samples, such as single-use blood lancing devices (e.g., Tasso, Inc., Seattle, WA) designed to obtain microliter capillary whole blood samples.

[0154] Dried Blood Spot (DBS) Dried blood spot (DBS) is a form of biological sample collection in which a blood sample is soaked and dried on filter paper. DBS typically involves the deposition of a small volume of capillary or venous blood onto a dedicated paper card. Compared to whole blood and plasma samples, their advantages lie in the fact that sample collection is easier and the logistical aspects associated with sample storage and transportation can be relatively limited without the need for refrigeration or dry ice, respectively. Wagner et al., Mass Spectrometry Reviews, 2016, 35, 361-438.

[0155] DBS typically consists of the deposition of a few drops of capillary blood obtained by heel or finger prick onto a card-type filter paper (also known as a Guthrie card). The sample is simply dried without any other processing. Chemically, the analytes are adsorbed along with the blood components onto a solid cellulose-based matrix. Compared to traditional venipuncture, a much smaller volume is required; blood collection is simple, noninvasive, and inexpensive; there is minimal risk of bacterial contamination or hemolysis; and DBS can be stored for long periods with little analyte degradation, which facilitates transportation due to sample stability. DBS sample collection requires minimal sample volumes, e.g., approximately 10-100, 20-80, or 30-70 microliters per spot. Therefore, DBS is appropriate when the blood volume to be collected is limited, such as in neonates, children, or critically ill patients.

[0156] Paper cards specifically for DBS are commercially available from several manufacturers and can be divided into two groups: raw paper and chemically modified paper. Raw paper consists of pure cellulose and can be manufactured from 100% pure cotton linters. Modified paper includes cellulose modified with various proprietary chemicals. These include Whatman (now part of GE Healthcare) FTA, FTA Elute, FTA DMPK-A, Whatman FTA DMPK-B (Majumdar & Howard, 2011), and Macherey Nagel NucleoCard (Moeller et al., 2012). FTA DMPK-A is impregnated with sodium dodecyl sulfate (SDS, <5%) and tris(hydroxymethyl)aminomethane (<5%), while FTA DMPK-B is impregnated with guanidine thiocyanate (30–50%). Alternatively, raw paper can be impregnated with chemicals by dipping it in a solution and allowing it to dry before use.

[0157] Blood collector cards, dried blood spot (DBS) technology, or HemaSpot™ devices, such as the HemaSpot™-HF device, can be used. For example, the HemaSpot™ HF device uses a fingerstick to collect and dry blood within a protective cartridge. For example, EBF blood spot collection cards, Eastern Business Forms, Inc., Mauldin, SC, such as 5-spot blood cards, or generic multi-part cards can be used, with each circle holding up to approximately 75-80 microliters of sample. Once dried, the sample is stable at room temperature and can be safely and easily transported to a laboratory for analysis.

[0158] Volumetric absorption microsampling Alternatively, a volumetric absorption microsampling (VAMS™) device can be utilized to obtain the blood sample. VAMS™ small volume collection devices, such as the Mitra® cartridge (Neoteryx, LLC), are commercially available. VAMS™ devices are handheld devices with a hydrophilic polymer tip connected to a plastic handle that, upon contact with the blood surface, draws up a fixed volume (approximately 10, 20, or 30 microliters) by capillary action. VAMS effectively provides for the absorption of a fixed volume of blood, regardless of hematocrit.

[0159] Volumetric absorption microsampling may utilize small volume sample collection, for example, sample volumes as low as 10, 20, or 30 microliters or more of blood sample, to draw a fixed volume of capillary blood, venous blood, or serum sample by capillary action.

[0160] Any suitable form of extraction may be utilized as known in the art. Analysis may be performed according to any suitable means, for example, LC-MS / MS.

[0161] DBS or VAMS blood samples can be eluted or extracted by any suitable means. DBS punch samples or VAMS chips can be exposed to an extraction solution to solubilize the analytes. The punch sample or VAMS chip can optionally be pre-soaked in water. The extraction solution can be, for example, water, acetonitrile, methanol, methanol-acetonitrile, methanol-water-formic acid, methanol-water (e.g., 90% aqueous MeOH; 4:1 v / v), or CHCl3 / MeOH (e.g., 2:1 v / v) at room temperature, e.g., about 25°C, for 30 minutes or more without stirring. Optionally, the punch sample in the extraction solution can be vortexed, sonicated, incubated, and centrifuged. The supernatant can be dried in a freeze dryer. The dried sample can be dissolved in or extracted with an extraction solution, diluted in a mobile phase buffer (e.g., acetonitrile-water-formic acid; e.g., 5:95:0.1, v / v), and transferred to a sample vial or multi-well format for any appropriate analytical method, including, for example, LC-MS / MS.

[0162] The following abbreviations are used in this disclosure: 13C-CA = carbon-13-labeled cholate; AIC = Akaike information criterion; BMI = body mass index; CI = confidence interval; CLD = chronic liver disease; CM = compartment model; d4-CA = deuterium-labeled cholate; DSI = disease severity index; ER = extraction fraction; HCV = hepatitis C virus; HFR = hepatic filtration fraction; HR = hepatic reserve; ICC = intraclass correlation coefficient; IV = intravenous; LC-MS = liquid chromatography-mass spectrometry; LC-MS / MS = liquid chromatography-tandem mass spectrometry. MM = minimal model; MMvd = minimal model based on volume of distribution; MSE = mean square error; NAFLD = non-alcoholic fatty liver disease; NASH = non-alcoholic steatohepatitis; SLD = fatty liver disease; MetALD = metabolic dysfunction-associated alcoholic liver disease; MASLD = metabolic dysfunction-associated fatty liver disease; MASH = metabolic dysfunction-associated steatohepatitis; TBV = total blood volume.

[0163] Compartmental model and non-compartmental analysis There are two main approaches that can be used to describe the pharmacokinetics (PK) of an administered compound: compartmental models and noncompartmental analysis. Compartmental models divide the body into a series of one or more, two or more, or three or more compartments of different volumes and are described by a set of kinetic equations that simulate the flow of drugs from one compartment to another. Compartmental models assume that each of the compartments is kinetically homogeneous and that the drug is instantly and uniformly distributed throughout the compartments. The mathematics of compartmental models typically involve a system of first-order ordinary differential equations with constant coefficients. While some compartmental models are descriptive and achieve an adequate fit to clearance data using two or three compartments, more realistic models that attempt to define the underlying physiological mechanisms can be developed using multicompartment systems. Noncompartmental analysis, on the other hand, as the name implies, makes no assumptions about body compartments and is considered an independent model. Noncompartmental analyses often use relatively simple algebraic equations to estimate summary PK parameters. As a result, noncompartmental methods require fewer assumptions than model-based approaches and are generally simpler, faster, and less expensive to develop compared to compartmental models.

[0164] Previously, noncompartmental analysis, hereafter referred to as the minimal model (MM), was used to characterize intravenous (IV) clearance by exponential fitting and oral clearance by cubic spline fitting (Everson, GT et al., Portal-systemic shunting in patients with fibrosis or cirrhosis due to chronic hepatitis C: the minimal model for measuring cholate clearances and shunt. Alimentary Pharmacology & Therapeutics, 2007. 26(3):401-410). More recently, the present inventors developed a compartmental model of the cholate SHUNT test that allowed for determination of anatomical shunt and hepatic extraction and improved the intraindividual reproducibility of SHUNT test measurements (McRae, MP et al., Compartmental model describing the physiological basis for the HepQuant SHUNT test. Translational Research).

[0165] This disclosure uses both compartmental and non-compartmental methods (i.e., DuO and TRIO) to estimate systemic and portal clearance from a reduced set of inputs and compares the results to previous cholate SHUNT liver function test measurements.

[0166] Artificial Intelligence (AI) Methods for Clearance Function Fitting An alternative approach to traditional pharmacokinetic analysis is to train AI algorithms to perform clearance curve fitting. AI methods, such as neural networks, can generalize nonlinear relationships between inputs and outputs. Such tools could be trained using the SHUNT study data (i.e., data collected at a sampling resolution of five oral and five intravenous time points) to estimate hepatic portal and systemic clearance functions (i.e., MM curves) using only a limited number of inputs (e.g., two oral and one intravenous time point). This current study investigates the feasibility of AI-based function estimators and nonlinear regressions, AI DuO and AI TRIO, to estimate systemic and portal clearance from a reduced set of inputs.

[0167] DuO cholate liver function test development A compartment model (CM) was developed to fit the portal clearance curve from the DuO cholate study. The compartment model is defined by the volume of distribution and the rate of transfer between volumes to estimate parameters not defined by noncompartmental analysis. The CM describes the transfer of cholate between the systemic, portal, and hepatic compartments, with assumptions from measured or literature-derived values ​​and unknown parameters estimated by nonlinear least-squares regression. Two versions of the DuO cholate study were developed.

[0168] The DuO cholate test estimates portal vein clearance using either one oral cholate dose and two blood draws (DuO v1.0, FIG. 1A) or two oral cholate doses and one blood draw (DuO v2.0, FIG. 1B). Both DuO test versions involve acquiring two distinct orally administered cholate concentration time points. Both DuO test versions include the same analytical methods.

[0169] Description of the DuO compartment model The flow between the systemic (S), portal (P), and hepatic (L) compartments is described by a system of first-order ordinary differential equations (Equations 1A-3), where q is the intercompartmental flow rate, V is the compartment volume, C is the concentration of cholate in the compartment, and Cl H is hepatic clearance.

[0170]

number

number

number

[0171] The following sections describe various aspects of the compartmental model.

[0172] Compartment Volume Total blood volume (TBV) was calculated based on the blood volume and body mass index (BMI, kg / m) across the full range of body weight (BW), including obese (BMI 30–40) and morbidly obese (BMI > 40) subjects. 2 ) is calculated using equation 4, which takes into account the nonlinear relationship between [8].

[0173]

number

[0174] In this equation, 22 is the BMI value corresponding to the ideal body weight, and 0.07 is the indexed blood volume (L kg ) for a subject with a BMI of 22. -1 )

[0175] Whole blood is composed of approximately 55% plasma and 45% hematocrit. [9] Because the SHUNT test uses serum sampling, any cholate distributed in the hematocrit is not measured. The plasma fraction (fplasma ) was used to adjust the volume according to the fraction of whole blood without red blood cells (i.e., 1-hematocrit) to approximate the serum sample collection used in the cholate SHUNT test. The total body compartment volume (V S ) is applied to the TBV as shown in Equation 4A. plasma This is the total plasma volume estimated by multiplying by 1, which is the total body compartment representing the total plasma volume from which the cholate measurement is derived. V S =TBV·(1-Hct) Equation 4A

[0176] It is estimated that the liver receives 25% of the total cardiac output [10, 11] and that the splanchnic organs contain 25% of the total blood volume at rest

[12] ; therefore, the volume of the hypothetical portal venous compartment (V) is 25%, as shown in Equation 4B. P ) was set to 25% of the total body volume. V P =0.25 V S Equation 4B

[0177] Liver compartment volume (V L To calculate the liver volume (22.46 ± 1.98 mL kg ), weight-corrected liver volumes from 13 ultrasound and computed tomography scan studies

[13] were averaged (22.46 ± 1.98 mL kg ). -1 ), BW and liver tissue density (d L ) to obtain the liver mass (w L ) was obtained. 1.06 g mL -1 The liver tissue density is 1.04 g mL -1

[14] and 1.08 g mL -1 The liver density was derived from the average of two studies estimating liver density

[15] . Liver blood volume ranges from 25 to 30 mL per 100 g liver mass [10, 16]. The average of this range (0.275 mL g -1 ) to calculate the volume of the liver compartment, V L was estimated by Equation 5A. V L =(0.275 22.46 BW dL f plasma ) / 1000 Equation 5A

[0178] Flow Parameters

[0179] The system of differential equations is S ), portal vein (C P ), and liver (C L ) describes the C-CA and d-CA concentrations in the compartment (Equations 1-3). Total liver flow rate (Q L ) is the hepatic artery (q SL ) and portal vein (q PL ) inflows (Equation 6A). Q L =q SL +q PL Equation 6A Liver mass w L Using previous estimates for the total liver flow velocity (Q L,init ) is approximately 1L -1 ·kg -1 and liver wet mass [10, 17].

[0180] Visceral circulation (q SP ) is defined as the blood flow to the abdominal gastrointestinal organs, including the stomach, liver, spleen, pancreas, and intestine. Various mechanisms for splanchnic blood flow control have been proposed, in which the splanchnic vascular bed functions with autoregulation to maintain constant blood flow over a range of perfusion pressures

[18] . For the purposes of this model, q SP was assumed to be proportional to the total cardiac output. The splanchnic circulation is proportional to the total hepatic inflow (Q L ) accounts for approximately 75% of

[10] , q SP Q L is defined as a constant flow rate at 75% of Q L is 25% of the total cardiac output.

[0181] The liver's intrinsic blood supply comes from both the hepatic artery and the portal vein. The hepatic arterial inflow (q SL) accounts for approximately 25% of the total inflow to the liver

[10] , is highly adaptive

[19] , and is able to compensate for changes in portal vein flow, with studies suggesting that 25%–60% of the reduced portal vein flow can be buffered by the hepatic artery [20, 21]. To simplify the model, q SL Q L is defined as 25% of Q L is 25% of the total cardiac output.

[0182] In healthy individuals, the portal venous inflow to the liver (q PL ) is the visceral artery flow velocity (q SP However, in the presence of collateral circulation (e.g., portosystemic shunt or esophageal-gastric varices), the portal inflow is equal to the shunt flow (q PS ) to simplify the model for the purpose of fitting a generalizable oral clearance curve to limited data points, no shunt flow rate was estimated (i.e., q PS was excluded from the model). Instead, the effect of shunting is captured in the estimation of the factor (F), which is related to absolute bioavailability and is described in more detail in the Oral Dose Administration section below.

[0183] Finally, the total hepatic venous return to the systemic circulation (q LS ) is the total inflow to the liver Q L is equal to.

[0184] liver extraction The hepatic extraction fraction (ER) is defined as the fraction of a drug entering the liver that is irreversibly eliminated during its first pass through the liver [22, 23]. Drugs with a high ER are rapidly eliminated by the liver, and clearance is primarily dependent on hepatic blood flow. Hepatic uptake of bile acids is exceptionally efficient, with extraction rates ranging from 50 to 90%, depending on the bile acid structure

[24] . Gilmore and Thompson studied the clearance of cholate in 14 human subjects and found a mean (standard deviation) extraction rate of 77.0% (7.5%)

[25] . Similarly, O'Maille, Richards, and Short measured an extraction rate of 79.0% (8.0%) in dogs

[26] . For cholate, an extraction rate greater than 0.7 is considered high

[27] ; therefore, clearance is primarily driven by hepatic flow. Because the concentration of cholate in systemic venous plasma is relatively low, and because it is extensively bound to albumin, the amount of cholate excreted in urine is negligible

[24] , and therefore it was assumed that administered cholate would be completely cleared by the liver.

[0185] Free cholate is removed from the liver with total hepatic clearance defined as hepatic inflow × extraction ratio [22, 23]. Due to the high extraction ratio of cholate (ER > 0.7) and relatively constant intrinsic hepatocyte clearance across the spectrum of liver disease, it is hypothesized that differences in cholate clearance in CLD are primarily due to changes in flux into the liver. In the DuO compartment model, hepatic clearance Cl H can be calculated according to Equation 7A.

[0186] Cl H =Q L ·ER Equation 7A

[0187] Here, ER was determined through parameter estimation using an initial guess of 0.7.

[0188] Effect of albumin It is important to note that the DuO cholate test measures the flow-dependent and highly efficient uptake of cholate by the liver, not its metabolism by hepatocytes. Hepatic uptake of organic anions has previously been explained according to the traditional "free drug" hypothesis, in which the concentration of free ligand controls the rate of hepatic uptake [28, 29]. However, despite their highly efficient hepatic uptake, organic anions have a strong affinity for binding to serum albumin. An alternative "albumin-mediated" uptake model may explain the highly efficient hepatic uptake of cholate [30, 31].

[0189] In the TRIO cholate test, intravenously administered identifiable cholate (e.g., 13C-CA) is pre-bound to albumin prior to administration to inhibit cell / tissue binding, ensure its retention in the vascular space, and promote efficient uptake by hepatocytes. For the oral identifiable cholate (e.g., d4-CA) dose in the DuO cholate test, it is assumed that the orally administered identifiable cholate (e.g., d4-CA) is extensively bound to albumin upon intestinal absorption. While a portion of the orally administered identifiable cholate (e.g., d4-CA) dose may be exposed to binding by red blood cells and extravascular tissues, for the sake of simplicity of the compartmental model, it is assumed that the administered dose enters and remains entirely within the vascular space and remains bound to albumin for the duration of the test until hepatic extraction.

[0190] Oral dose administration

[0191] Oral dose administration has been modeled via a flexible transport model [32, 33], which has been demonstrated to account very well for the absorption delay observed in oral drug administration. The transport model is designed to transport the oral dose (D) through a series of n non-integer virtual transport compartments to simulate drug absorption delay and account for first-pass extraction. PO ) was adapted to account for the passage of d4-CA into the systemic circulation. Equation 8A is the rate of change of the amount of d4-CA entering the systemic circulation (dA d4 / dt).

[0192]

number

number

[0193] where t is time in minutes; k TR is the intercompartmental transfer rate constant (Equation 9A); MTT is an estimated parameter representing the mean transit time of d4-CA molecules into the systemic circulation via intestinal absorption (initial estimate of 30 min); F is an estimated parameter that scales the oral clearance curve and is related to first-pass bioavailability (initial estimate of 0.20).

[0194] DuO method for calculating liver disease index

[0195] The following metrics were measured by the DuO cholate liver function test and have previously demonstrated association with liver function: Oral area under the curve (AUC Oral ) is measured by first simulating the complete oral clearance curve using a compartmental model and calculating the area using trapezoidal numerical integration. Portal hepatic filtration rate (HFR P ) is the portal vein clearance adjusted for subject body weight (BW) calculated by Equation 10A.

[0196]

number

[0197] The following metrics are estimated by the cholate DuO test and have previously demonstrated association with liver function: ·Area under the IV curve (AUC IV) is estimated via a linear regression model (Equation 11A). AUC IV =β0+β BW BW+β PO,20 C PO,20 +β PO,60 C PO,60 +β HFR,P HFR P Equation 11A This linear model is compared with the AUC from SHUNT-V. IV The data was used for training, resulting in the regression coefficients listed in Table 1B. To protect against overfitting, a linear model was trained using a 5-fold cross-validation procedure by partitioning the data into folds and estimating the accuracy for each fold. The predictors considered in the model were AUC Oral and V d In addition to the above, we included those listed in Table 1B. Predicted AUC IV and the AUC calculated from the minimum model IV The best-performing model and subset of model parameters that minimized the root mean square error (RMSE) between

[0198] [Table 2]

[0199] Whole body hepatic filtration rate (HFR) S ) is the estimated systemic clearance adjusted for body weight (Equation 12), where IV dose (D IV ) is assumed to be half the administered oral dose.

[0200]

number

[0201] SHUNT is the estimated absolute bioavailability of orally administered distinguishable cholate (e.g., d4-CA) (Equation 13).

[0202]

number

[0203] The DSI is a score that indicates overall liver function, including both portal and systemic HFR. The DSI is intended to convey unique information about the clinical stage of fibrosis and cirrhosis, and modeling these outputs for the prediction of clinical outcomes generated the DSI in Equation 14 [6, 34-36]. Here, HFR P,max and HFR S,max is the upper limit of clearance relative to healthy controls, and A is a coefficient for scaling the DSI from 0 to 50.

[0204]

number

[0205] Indexed hepatic reserve (HR indexed ) is a numerical index of overall liver health with values ​​ranging from 0 to 100 (Equation 15). Here, HR indexed HR from 100 to 0 indexed HFR indexed to lean controls -1 standard deviation by a constant A to scale P and HFR S (HFR P,lean and HFR S,lean ) is used.

[0206]

number

[0207] Algebraic hepatic reserve (HR algebraic ) is a numerical index of overall liver health with values ​​between 0 and 50 (Equation 15A). HR algebraic =100-2·DSI Equation 15A

[0208] TRIO Cholate Liver Function Test Development The TRIO cholate test is similar to the DuO cholate test, except for the addition of a single IV dose of identifiable cholate (e.g., 13C-CA) and its measurement over 20 minutes. This addition of data point allows for a more accurate estimation of the systemic clearance curve, thereby improving the AUC Oral and AUC IV It allows for quantification of test parameters that require accurate measurement of both, such as whole-body HFR, DSI, SHUNT, and HR.

[0209] The TRIO test quantifies portal vein HFR, systemic HFR, DSI, SHUNT%, HR, and RCA-20.

[0210] Two versions of the TRIO cholate liver function test have been developed. The TRIO test estimates both portal and systemic clearance using either one oral dose + one IV dose + two blood draws (TRIO V1.0, also known as SHUNT 2.0, Figure 2A) or two oral doses + one IV dose + one blood draw (TRIO V2.0, also known as SHUNT 3.0, Figure 2B). Both versions of the TRIO test use the same analytical method.

[0211] A noncompartmental analysis method was developed that involved exponential fitting of systemic cholate clearance using only 20-minute intravenous discriminative (e.g., 13C-CA) concentration time points. The exponential fitting divides the curve into three sections or phases: fast, moderate, and slow (0-20 min, 20-45 min, and 45-180 min for Y0, Y1, and Y2, respectively) (Figure 3).

[0212] TRIO analysis explained rapid phase The first phase of clearance, from 0 to 20 min, represents the rapid distribution phase. To estimate the initial C-CA concentration, the volume of distribution (V) in L per kg body weight was calculated. d ) were compared with blood volume and body mass index (BMI, kg / m ) across the full range of body weights, including obese (BMI 30–40) and morbidly obese (BMI > 40) subjects. 2) was first calculated using Equation 16A, which takes into account the nonlinear relationship between

[0213]

number

[0214] In this equation, 22 is the BMI value corresponding to the ideal body weight, and 0.07 is the indexed blood volume (L kg ) for a subject with a BMI of 22. -1 ) Then, the initial 13C-CA concentration after the dose was measured by dividing the dose by V d × body weight (BW) (Equation 17).

[0215]

number

[0216] Finally, the rate of elimination in the fast phase is k fast is defined by (Equation 18).

[0217]

number

[0218] proper speed phase The second phase of clearance, occurring over 20-45 minutes, is characterized by an elimination rate of k mod represents the optimal elimination phase, estimated using a linear model (Equation 19). k mod =β 0,mod +β kfast ·k fast +β IV,20 C IV,20 +β PO,20,mod C PO,20 +β PO,60,mod C PO,60 Equation 19

[0219] This linear model was trained using the systemic clearance data from the SHUNT-V study, resulting in the regression coefficients listed in Table 2. To protect against overfitting, the linear model was trained using a 5-fold cross-validation procedure by partitioning the data into folds and estimating precision for each fold. Predictors considered in the model included those listed in Table 2, in addition to body weight and IV dose. The predicted k mod and k calculated from the minimum model mod The best-performing model and subset of model parameters that minimized the root mean square error (RMSE) between

[0220] [Table 3]

[0221] slow phase The terminal phase of clearance over a period of 45 minutes is characterized by an elimination rate of k slow is 0.018 minutes -1 Figure 1 represents the slow elimination phase, which was set to the mean value of SHUNT-V subjects who were

[0222] Exponential Fitting The exponential equation governing the systemic concentration of 13C-CA over time is:

number

number

number

[0223] where t is time (0-20 min, 20-45 min, and 45-180 min for Y0, Y1, and Y2, respectively), C0 is the initial concentration of 13C-CA, and C20 is the measured 20-minute concentration of 13C-CA, and C 45 is the estimated 45-minute concentration of 13C-CA. The area under each of the three exponential curve fits was calculated by trapezoidal numerical integration, and AUC IV are summed to estimate

[0224] TRIO method for calculating disease index The following metrics are measured by the cholate TRIO test and have previously demonstrated association with liver function: Oral area under the curve (AUC Oral ) - Same as DuO Portal hepatic filtration rate (HFR P ) - Same as DuO ·Area under the IV curve (AUC IV ) is measured by trapezoidal numerical integration of the exponential fitting from TRIO. ·Whole body hepatic filtration rate (HFR) S ) is the measured systemic clearance adjusted for body weight using the same equation as DuO (Equation 12), however, the IV dose (D IV ) is the actual dose and AUC IV is measured as described above. SHUNT uses the same equation as DuO (Equation 13), but instead measures the AUC IV It is calculated using the value. DSI uses the same equation as DuO (Equation 14), but instead measures the AUC IV It is calculated using the value. Hepatic reserve (HR) uses the same equation as DuO (Equation 15), but instead measures the AUC IV It is calculated using the value.

[0225] Cholate AI Test Method Artificial intelligence (AI), implemented using methods such as neural networks, allows for the learning and fitting of complex functions using a limited number of inputs. Function fitting is the process of training a neural network on a set of inputs to generate a related set of target outputs. Here, a neural network is constructed with a desired network topology and a learning algorithm and trained using a set of training data. After the network has fitted the data, it forms a generalization of the input-output relationship. This trained network is then used to predict outputs for future data not used in training.

[0226] In this analysis, a function fitting neural network was used to predict complete oral and IV clearance curves using data from the cholate DuO and TRIO studies (i.e., cholate AI DuO and AI TRIO). Although this analysis focuses on the application of a function fitting neural network, similar results could be achieved using alternative methods for function approximation and nonlinear regression, including nonparametric statistics (NNS), deep learning (i.e., deep neural networks), generative adversarial networks (GANs), random forest regression, ensemble methods, and / or any combination of interpolation, extrapolation, nonlinear regression, and curve fitting methods.

[0227] AI analysis can include using function fitting neural networks to predict complete oral and IV clearance curves using data from the DuO and TRIO liver function tests provided herein.

[0228] Cholate AI DuO test method The goal of the AI ​​DuO method is to predict both oral and IV clearance curves using only orally administered identifiable cholate (e.g., d4-CA) clearance data from DuO studies. The complete list of inputs to the neural network is: (i.) 20-minute oral cholate (e.g., d4-CA) concentration; (ii.) 60-minute oral cholate (e.g., d4-CA or d5-CA) concentration; (iii.) Estimated V d (Equation 16A); (iv.) BMI; and (v.) Hematocrit Contains a 5x1 array of

[0229] The neural network outputs selected points from the oral and IV curves estimated by the MM (i.e., 5-minute increments from 0 to 180 minutes, resulting in 37 time points for both the oral and IV curves, rearranged into a 74 × 1 array). The network was configured for a regression task and consisted of a two-layer feedforward network with a sigmoidal transformation function in the hidden layer and a linear transformation function in the output layer (Figure 4). The hidden layer size was optimized with respect to performance (mean squared error) on the test set.

[0230] The training, validation, and test data consisted of a total of 542 subjects (N=217 from the HALT-C study, N=275 from the SHUNT-V study, and N=50 healthy with lean and overweight body mass index). The training data was divided into training (70%), validation (15%), and test sets (15%). The training algorithm was Levenberg-Marquardt backpropagation; however, many alternative training algorithms could be used (Bayesian regularization, BFGS quasi-Newton methods, resilient backpropagation, scaled conjugate gradients, conjugate gradients with Powell / Beale restart, Fletcher-Power conjugate gradients, Polak-Ribiere conjugate gradients, one-step secant methods, variable learning rate gradient descent, gradient descent with momentum, and gradient descent, etc. [Hagan, M.T., H.B. Demuth, and M.H. Beale, Neural Network Design, Boston, MA: PWS Publishing, 1996, Chapters 11 and 12]). Model performance was externally validated using data from a reproducibility study (see the Reproducibility Analysis section below for results).

[0231] Cholate AI TRIO Test Method The goal of the AI ​​TRIO method is the same as AI DuO, to predict both oral and IV clearance curves; however, it uses two oral d4-CA measurements and one 13C-CA measurement from the TRIO study. The complete list of inputs to the neural network is: (i.) 20-minute oral discernible cholate (e.g., d4-CA) concentration; (ii.) 60-minute oral discernible cholate (e.g., d4-CA or d5-CA) concentration; (iii.) 20-minute intravenous discernible cholate (e.g., 13C-CA) concentration; (iv.) Estimated V d (Equation 16A); and (v.) V d Estimated initial concentration of intravenous discernible cholate (e.g., 13C-CA) based on Contains a 5x1 array of

[0232] The AI ​​TRIO network has the same architecture (Figure 4) and training method as AI DuO.

[0233] Cholate AI method for calculating liver disease index The cholate test AI DuO and AI TRIO analyses generate both oral and IV clearance curves. AUC IV and AUC Oral is measured by trapezoidal numerical integration and the residual test parameters are calculated: HFR P (Equation 10A);HFR S (Equation 12); SHUNT (Equation 13); DSI (Equation 14); and HR (Equation 15).

[0234] Minimal model based on volume of distribution (MM) Vd ) Previously, a noncompartmental analysis of the cholate SHUNT liver function test, hereafter referred to as the minimal model (MM), was used to characterize intravenous (IV) clearance by exponential fitting and oral clearance by cubic spline fitting (Everson et al., Alimentary Pharmacology & Therapeutics, 2007. 26(3):401-410). This method used the slope of the 5- and 20-minute IV log-linear regression to estimate the concentration of C-CA at 0 minutes.

[0235] The initial clearance of IV-administered 13C-CA from the systemic circulation is a rapid exponential decrease in concentration. Therefore, estimation of initial concentrations based on the 5-minute time point may be sensitive to variability in study performance (e.g., timing of dose administration and 5-minute blood sample collection, single-arm vs. double-arm catheter administration / collection, residual cholate in the catheter lines, etc.). In other words, small variations in the timing of measurements and 5-minute 13C-CA concentrations can affect the IV area under the curve (AUC IV), and therefore will lead to significant changes in the dependent cholate test parameters (whole body HFR, SHUNT, DSI, HR).

[0236] A new version of the cholate minimum model analysis (MM) based on the volume of distribution, in which the initial intravenous discernible cholate (e.g., 13C-CA) concentration at 0 min is calculated using subject physical characteristics (weight and BMI) as opposed to estimation based on the slope of the 5- and 20-min IV log-linear regression. Vd The analytical method (SHUNT V1.1 test) is provided herein. d is first estimated by Equation 16A, followed by V d The initial concentration is estimated by dividing the IV dose by the body weight × 1. This estimated initial concentration value is then used in the original MM (SHUNT V1.0) equation to construct the IV clearance curve.

[0237] Reproducibility analysis data Data from a previous study of cholate test reproducibility were retrospectively analyzed. The study consisted of 48 subjects from three groups: controls (N = 16), NASH (N = 16), and HCV (N = 16). Controls were healthy individuals with no history of liver disease and normal standard blood tests. NASH was diagnosed based on risk factors (obesity, diabetes, metabolic syndrome), negative tests for other liver diseases, and fibrosis staging by liver biopsy or transient elastography. HCV was diagnosed based on a history of positive HCV by nucleic acid testing and METAVIR fibrosis staging by liver biopsy. Three repeat cholate SHUNT tests were performed on three separate days within 30 days. The original study provides further details regarding recruitment and study conduct (Burton, JR et al., Translational Research, 2021, 233:5-15).

[0238] statistical analysis Test parameters were measured or estimated for all six methods (DuO, TRIO, AI DuO, AI TRIO, MM, and MM Vd ). Reproducibility was assessed through intraclass correlation coefficients (ICCs) calculated for indices of liver disease for CM and MM, and for key PK parameters for CM. ICCs were single-rater / measure two-way mixed-effects models for absolute agreement between measurements [38, 39], with a one-sided test for a lower acceptance limit (ICC > 0.7), previously defined by a reduction in precision of less than 5%

[37] . Correlation plots were analyzed using Deming regression fitting

[40] to assess systematic differences and the coefficient of determination (R) to assess the association between the two methods. 2 ) were used to measure the MM and MM for key cholate test parameters. Vd Each method is compared by:

[0239] Results and Discussion Cholate 2nd generation (2 nd The second-generation (V) tests (DuO, TRIO, AI DuO, and AI TRIO) correlated well with previously validated liver function test parameters. All second-generation methods yielded similar or better reliability in terms of CV and ICC compared with the MM method. For all test parameters, the second-generation tests were significantly more reliable than the original MM. d Based on MM(MM Vd ) correlated well with (V d (Except for portal vein HFR, which was independent of vasopressin).

[0240] Portal vein HFR MM method (upper panel) and MM Vd Portal vein hepatic filtration rate (HFR) in second-generation cholate trials (DuO, TRIO, AI DuO, AI TRIO) and first-generation SHUNT trial results analyzed by the method (lower panel). P Six graphs illustrating the correlation of ) are shown in Figure 5A. The TRIO graph is the same as that of DuO and is not shown.

[0241] In the case of portal vein HFR (Figure 5A), information about systemic clearance is not used, and the calculations, therefore, for the DuO and TRIO methods are equivalent. Similarly, the volume of distribution has no effect on the fitting function of the oral clearance curve, and as expected, the MM and MM Vd HFR with the same law P The value was obtained.

[0242] Table 3 shows the HFR by analytical method as measured by the coefficient of variation (CV) and intraclass correlation coefficient (ICC) for all subjects (N=48). P Demonstrates reproducibility.

[0243] [Table 4]

[0244] Table 4 (Fig. 5B) shows the HFR by analytical method as measured by the coefficient of variation (CV) and intraclass correlation coefficient (ICC) for patient groups (control, N=16; NASH, N=16; HCV, N=16). P Demonstrates reproducibility.

[0245] All six methods yielded similar reliability in terms of CV and ICC, with slightly better reproducibility measured for the AI ​​DuO and AI TRIO methods across the overall study population (Table 3) and within patient groups (Figure 5B, Table 4). These results suggest that portal vein HFR can be measured with acceptable precision and reliability using the dual oral (DuO) method, using either a compartmental model or AI analysis.

[0246] Whole body HFR MM method and MM Vd Whole-body HFR (HFR) in the results of second-generation cholate trials (DuO, TRIO, AI DuO, AI TRIO) and first-generation SHUNT trials analyzed by the method S ) are shown in Figure 6A.

[0247] Regarding whole-body HFR (Figure 6A), the addition of 20-minute IV data points in TRIO resulted in a substantial improvement in correlation with MM (HFR of 0.61 and 0.86 for DuO and TRIO, respectively). S R 2 ).

[0248] Table 5 shows the HFR by analytical method as measured by the coefficient of variation (CV) and intraclass correlation coefficient (ICC) for all subjects (N=48). S Demonstrates reproducibility.

[0249] [Table 5]

[0250] Table 6 (Fig. 6B) shows the HFR by analytical method as measured by the coefficient of variation (CV) and intraclass correlation coefficient (ICC) for patient groups including NASH and HCV (control, N=16; NASH, N=16; HCV, N=16). S Demonstrates reproducibility.

[0251] Additionally, all methods showed a significant improvement in MM compared to MM. Vd The best performing models by far were TRIO, MM, and MM, respectively. Vd R of 0.86 and 0.97 for 2 Regarding intra-individual reproducibility, the second-generation method had significantly lower CV and better ICC compared to MM (Table 5 and Table 6, Figure 6B). Although DuO and AI DuO had the lowest variability, these methods were less precise than the TRIO and AI TRIO methods, which included 20-minute IV data. The TRIO method had significantly lower variability compared to the MM method (CV of 5.7% and 11%; ICC of 0.91 and 0.82 for TRIO and MM, respectively). These results suggest that accurate and reliable systemic clearance curves can be generated using a single 20-minute time point and an exponential fitting approach.

[0252] SHUNT SHUNT is AUC IV AUC for Oral Since the ratio of systemic and portal clearance is a ratio, an accurate and reliable estimation of both systemic and portal clearance is desirable.

[0253] MM method and MM Vd Eight graphs illustrating the correlation of SHUNT% among cholate second-generation tests (DuO, TRIO, AI DuO, AI TRIO) and first-generation cholate SHUNT test results analyzed by the method are shown in Figure 7A.

[0254] Similar to whole-body HFR, an improvement in correlation was found for SHUNT when IV 20-minute data points were added (FIG. 7A) (SHUNT R of 0.67 and 0.84 for DuO and TRIO, respectively). 2 ). Additionally, all methods performed significantly better than MM compared to MM. Vd showed a significantly good correlation with

[0255] By far the best performing models are the TRIO, MM and MM Vd R of 0.84 and 0.96 2 Regarding intra-individual reproducibility, the second-generation method had significantly lower CV and better ICC compared with MM (Tables 7 and 8).

[0256] Table 7 shows the SHUNT reproducibility by analytical method as measured by the coefficient of variation (CV) and intraclass correlation coefficient (ICC) for all subjects (N=48).

[0257] [Table 6]

[0258] Table 8 (Figure 7B) shows the SHUNT reproducibility by analytical method as measured by the coefficient of variation (CV) and intraclass correlation coefficient (ICC) for patient groups (control, N=16; NASH, N=16; HCV, N=16).

[0259] All second-generation methods had reliable measurements of SHUNT, but MM failed to achieve an ICC greater than 0.7 (p=0.3105). Again, DuO and AI DuO had the lowest variability, but these methods were less accurate than the TRIO and AI TRIO methods, which included 20-minute IV data. The TRIO method had lower variability compared to the MM method (CVs of 12.1% and 15.3% for TRIO and MM, respectively; ICCs of 0.79 and 0.73). These results further support the finding that accurate and reliable measurements of both systemic and portal vein clearance curves can be generated using the TRIO approach.

[0260] DSI and HR MM method and MM Vd Eight graphs illustrating the correlation of Disease Severity Index (DSI) among cholate second-generation studies (DuO, TRIO, AI DuO, AI TRIO) and first-generation SHUNT study results analyzed by the method are shown in Figure 8A.

[0261] Table 9 shows the DSI reproducibility by analytical method as measured by the coefficient of variation (CV) and intraclass correlation coefficient (ICC) for all subjects (N=48).

[0262] [Table 7]

[0263] Table 10 (Figure 8B) shows the DSI reproducibility by analytical method as measured by the coefficient of variation (CV) and intraclass correlation coefficient (ICC) for patient groups (control, N=16; NASH, N=16; HCV, N=16).

[0264] MM method and MM Vd Eight graphs illustrating the correlation of hepatic reserve (HR) among cholate second-generation studies (DuO, TRIO, AI DuO, AI TRIO) and first-generation SHUNT study results analyzed by the method are shown in Figure 9A.

[0265] Table 11 shows the HR reproducibility by analytical method as measured by the coefficient of variation (CV) and intraclass correlation coefficient (ICC) for all subjects (N=48).

[0266] [Table 8]

[0267] Table 12 (Figure 9B) shows the HR reproducibility by analytical method as measured by the coefficient of variation (CV) and intraclass correlation coefficient (ICC) for patient groups (control, N=16; NASH, N=16; HCV, N=16).

[0268] DSI (Fig. 8A) and HR (Fig. 9A) were calculated based on HFR. S and HFR P However, there was only a slight improvement in the correlation between MM and second-generation studies when IV data were added (DSI R of 0.94 and 0.97 for Duo and Trio, respectively). 2 HR R of 0.97 and 0.98 for Duo and Trio, respectively 2 ). Additionally, all methods performed significantly better than MM compared to MM. Vd showed a slightly better correlation with

[0269] Regarding the intra-individual reproducibility of DSI, the second-generation method had significantly lower CV and better ICC compared with MM (Tables 9 and 10). This effect was due to the HFR SThis is particularly noticeable in controls, which have relatively high variability (i.e., relatively high uncertainty in LC-MS measurements at low concentrations) due to heterogeneous variance of DSI. Because the HR index discards those with high HFR (i.e., values ​​exceeding the mean of lean controls minus one standard deviation), all methods had similar reproducibility characteristics for measuring HR. These results suggest that both DuO and TRIO provide acceptable precision and reliability in measuring DSI and HR compared with the MM approach.

[0270] SHUNT V2.0 and alternative DuO analysis methods This section describes exemplary SHUNT V2.0 and alternative DuO analysis methods. SHUNT V2.0 is a simplification of the SHUNT test that measures 13C-CA (intravenous dose) and d4-CA (oral dose) concentrations at 20 and 60 minutes. SHUNT V2.0 uses exponential fitting for systemic cholate clearance and compartmental analysis for portal venous cholate clearance, based on assumptions of liver flow and physiology. DuO measures only d4-CA concentrations at 20 and 60 minutes to determine portal venous cholate clearance using the same compartmental model. A detailed description of the compartmental model describing the compartmental model of the cholate SHUNT V1.0 test, which enabled determination of anatomical shunt and hepatic extraction and improved intra-individual reproducibility of SHUNT test measurements, is provided in McRae MP, Helmke SM, Burton JR, Jr., Everson GT. Compartmental model describing the physiological basis for the HepQuant SHUNT test. Transl Res. 2023;252:53-63, which is incorporated herein by reference in its entirety. The following sections highlight the key differences between the original model (for SHUNT V1.0) and two of the simplified models (SHUNT V2.0 and DuO).

[0271] Compartmental model of portal venous clearance

[0272] The flow between the systemic (S), portal (P), and hepatic (L) compartments is described by a system of first-order ordinary differential equations (Equations S1–S3), where q is the intercompartmental flow rate, V is the compartment volume, C is the concentration of cholate in the compartment, and Cl H is the hepatic clearance and D PO,rate is the rate of orally administered d4-cholate entering the portal compartment.

[0273]

number

number

number

[0274] The main difference between the SHUNT V1.0 compartment model and the simplified model is that the original model took cholate binding into account. It is important to note that the HepQuant study measures the flow-dependent and highly efficient uptake of cholate by the liver, not its metabolism by hepatocytes. In the HepQuant study, intravenously administered 13C-CA was prebound to albumin prior to administration to inhibit cell / tissue binding, ensure its residence in the vascular space, and promote efficient uptake by hepatocytes. Orally administered d4-CA was assumed to bind extensively to albumin during intestinal absorption. While a portion of the oral d4-CA dose may be exposed to binding by red blood cells and extravascular tissues, for the sake of simplicity of the compartment model, it was assumed that the administered dose entered and remained entirely within the vascular space, bound to albumin until hepatic extraction.

[0275] In healthy individuals, the portal venous inflow to the liver (q PL) is the visceral artery flow velocity (q SP However, in the presence of collateral circulation (e.g., portosystemic shunt or esophageal-gastric varices), the portal inflow is equal to the shunt flow (q PS ) to simplify the model for the purpose of fitting a generalizable oral clearance curve to a limited number of data points, the shunt flow rate was estimated by estimating a parameter (i.e., a constant between 0 and 1) representing the fraction of the splanchnic circulation that is shunted. The magnitude of the oral clearance curve was independently adjusted through estimation of a factor F related to absolute bioavailability.

[0276] The hepatic extraction fraction (ER) is defined as the fraction of drug entering the liver that is irreversibly removed during the first pass through the liver [29, 30]. Free cholate is cleared from the liver with total hepatic clearance defined as hepatic inflow × extraction fraction. Due to the high extraction fraction of cholate (ER > 0.7) and relatively constant intrinsic hepatocyte clearance across the spectrum of liver disease, it is hypothesized that differences in cholate clearance in CLD are primarily due to changes in flux into the liver. In a compartmental model, hepatic clearance Cl H was calculated according to equation S4.

[0277] Cl H =Q L ·ER Equation S4

[0278] In the simplified compartmental model, rather than including ER as an estimated parameter, the estimated parameters F and shunt flow fraction were both held constant at 0.75 and iteratively reduced by 5% when they were above 50%, thereby allowing the model to fit to data showing reduced hepatocyte extraction.

[0279] Oral dose administration was modeled via a flexible transport model (Savic et al., Implementation of a transit compartment model for describing drug absorption in pharmacokinetic studies. J Pharmacokinet Pharmacodyn. 2007;34:711-26; Wilkins et al., Population pharmacokinetics of rifampin in pulmonary tuberculosis patients, including a semimechanistic model to describe variable absorption. Antimicrob Agents Chemother. 2008;52:2138-48). Flexible transport models have been demonstrated to account very well for the absorption delay observed with oral drug administration. The transport model was modeled as a D 1000-1200 sigma-based transport model, which involves the transport of rifampin through a series of n non-integer virtual transport compartments to simulate drug absorption delay and account for first-pass extraction. PO Equation S5 is the rate of change of the amount of d4-CA entering the systemic circulation (D PO,rate ) is explained.

[0280]

number

number

[0281] where t is time in minutes; k TR is the intercompartmental transfer rate constant (Equation S6); MTT is an estimated parameter representing the mean transit time of d4-CA molecules into the systemic circulation via intestinal absorption (initial estimate of 30 minutes); F is an estimated parameter that scales the oral clearance curve and is related to first-pass bioavailability (initial estimate of 0.20). Equations S5 and S6 define the shape of the oral clearance curve.

[0282] Non-compartmental analysis of systemic clearance Non-compartmental analysis for SHUNT V2.0 involves exponential fitting of systemic cholate clearance using only the 20-minute C-CA concentration time point. The exponential fitting divides the curve into three sections or phases: fast, moderate, and slow.

[0283] The first phase of clearance, from 0 to 20 min, represents the rapid distribution phase. To estimate the initial C-CA concentration, the total blood volume (TBV) in liters per kg body weight was calculated as a function of blood volume and body mass index (BMI, kg / m) across the full range of body weights, including obese (BMI 30-40) and morbidly obese (BMI > 40) subjects. 2 ) was first calculated by equation S7, which takes into account the nonlinear relationship between blood volume and blood pressure (Lemmens et al., Estimating blood volume in obese and morbidly obese patients. Obes Surg. 2006;16:773-6).

[0284]

number

[0285] In equation S7, 22 is the BMI value corresponding to ideal body weight, and 0.07 is the indexed blood volume (L·kg ) for a subject with a BMI of 22. -1 ) The initial 13C-CA concentration after the dose was then measured. d × body weight (BW) (Equation S8).

[0286]

number

[0287] Finally, the rate of elimination in the fast phase is k fast (Equation S9), where C 20is the concentration at 20 minutes, and T 20 is the actual time recorded for the 20 minute sample.

[0288]

number

[0289] The second phase of clearance, occurring over 20 to 60 minutes, is characterized by an elimination rate of k mod represented the optimal elimination phase, defined by equation S10.

[0290]

number

[0291] The terminal phase of clearance over a period of more than 60 minutes is characterized by an elimination rate k slow is 0.0183 minutes -1 The exponential equation governing the systemic concentration of 13C-CA over time was:

[0292]

number

number

number

[0293] where t is time (0-20 min, 20-60 min, and 60-180 min for Y0, Y1, and Y2, respectively), C0 is the initial concentration of 13C-CA, and C 20 and C 60 are the measured 20-minute and 60-minute concentrations of 13C-CA, and T 20 and T 60are the actual 20- and 60-minute sampling times for 13C-CA. The area under each of the three exponential curve fits was calculated by trapezoidal numerical integration, and AUC IV are summed to estimate

[0294] Estimation of IV concentrations for DuO

[0295] For the oral-only simplified liver function test DuO, the derived IV 20- and 60-minute concentrations were estimated by Equation S16 and Equation S17.

[0296]

number

number

[0297] A linear model was trained using data from the SHUNT-V Study (ClinicalTrials.gov. The SHUNT-V Study for Varices. clinicaltrials.gov / ct2 / show / NCT03583996. Accessed July 20, 2022) (N=275) and healthy controls (N=50), resulting in the normalization constants and regression coefficients listed in Table 14. The AUC for DuO was then calculated. IV was calculated by the same method as SHUNT V2.0, substituting estimated 20- and 60-minute IV concentrations rather than measured values.

[0298] [Table 9]

[0299] Estimation of portal vein HFR and DSI for the STAT test STAT is simply the d4-CA concentration at 60 minutes adjusted for 75 kg body weight by the calculation ([d4-CA] x kg body weight / 75 kg). Values ​​derived from the STAT test can be used by themselves or in the estimation of portal vein HFR and DSI (Figure 13).

[0300] To estimate portal venous HFR, equation S14 was derived by fitting the parameters for an exponential function to the portal venous HFR calculated from SHUNT V1.0. HFR P,est =2.1308+54.4412e -6.0001·STAT +21.0936e -0.8578·STAT Equation S14

[0301] To estimate DSI, Equation S15 was derived by fitting parameters for a square root function to the DSI calculated from SHUNT V1.0.

[0302]

number

[0303] Use of Duo or Trio Liver Function Tests to Measure Therapeutic Efficacy Changes in portal circulation can be detected by portal HFR, SHUNT%, first-pass hepatic extraction of identifiable cholate, and DSI. Changes in one or more of these parameters can detect changes in portal circulation. The prior art cholate SHUNT test (SHUNT 1.0) using minimal model data analysis was able to detect treatment effects in clinical trials. Lawitz, E. et al., BI 685509 improves hepatic function in subjects with Child-Pugh A cirrhosis and a liver stiffness measurement of >15 kPa: Results from the HepQuant SHUNT test. Hepatology, 2021(74):1238A-1239A. The simpler cholate DuO and TRIO tests of the present invention can be used to detect the same treatment effects.

[0304] For example, the Duo and / or Trio methods of the invention provided herein can be used in combination with investigational or known drugs, such as non-selective beta-adrenergic blockers, ACE inhibitors, guanylate cyclase activators, thyroid hormone receptor beta agonists (THRs), and / or steroids. β agonists), cyclophilin inhibitors, or other vasoactive agents used to alter hepatic and portal blood flow and portosystemic shunting, or liver disease therapeutics that alter liver fat content, fibrosis, or hepatocellular function, or any other drug that may affect liver function that may affect steatosis. [Example]

[0305] Example 1. Simplified Cholate Test: Reproducibility and Diagnostic Performance Data for Predicting Large Esophageal Varices Example 1A. Simplified Cholate Test: SHUNT V1.1 and SHUNT 2.0 for Predicting Large Esophageal Varices Background and Objectives: The Cholate SHUNT Liver Function Test (Minimal Model, MM=SHUNT V1.0) uses stable isotope-labeled cholate administered both intravenously (C-CA) and orally (d4-CA) to quantify liver function and physiology from blood samples taken at 0, 5, 20, 45, 60, and 90 minutes relative to serum cholate. The MM Cholate SHUNT Test has been used in over 26 clinical trials and studies covering a wide range of etiologies and stages of liver disease and has compared favorably with other liver diagnostic tests. However, the MM Cholate SHUNT Test is sensitive to variability in the timing of 5-minute blood sample collection and difficulties in maintaining intravenous access. The purpose of this study was to enhance the MM Cholate SHUNT Test and its performance by simplifying the sample collection procedure and reducing test time. Herein, the MM Cholate SHUNT Test is used in over 26 clinical trials and studies covering a wide range of etiologies and stages of liver disease and has compared favorably with other liver diagnostic tests. However, the MM Cholate SHUNT Test is sensitive to variability in the timing of 5-minute blood sample collection and difficulties in maintaining intravenous access. The purpose of this study was to enhance the MM Cholate SHUNT Test and its performance by simplifying the sample collection procedure and reducing test time. Vd Two new versions of the cholate test were evaluated, including the SHUNT V1.1 test and the TRIO V1.0 (=SHUNT V2.0) test.

[0306] Methods: In the MM cholate SHUNT study, the volume of distribution (Vd) was calculated from the ln-linear regression of 5- and 20-minute 13C-CA concentrations against time. VdThe cholate test estimates Vd based on weight and height, eliminating the requirement for a 5-minute blood sample (Lemmens et al., 2006). The TRIO (v1.0) cholate test is based on a published compartmental analysis (McRae et al., 2022) and further simplifies sample collection requirements for two time points at 20 and 60 minutes. To compare reproducibility, the coefficient of variation (CV) and intraclass correlation coefficient (ICC) using a one-sided test with a lower acceptance limit of 0.7 were analyzed in a study of 16 controls, 16 NASH patients, and 16 HCV patients, each with three replicates performed on three separate days (Burton et al., 2021). Differences in the area under the receiver operating characteristic curve (AUROC) for predicting large esophageal varices (LEV) in hepatitis C (HCV) subjects (N=217) from the HALT-C study (Everson et al. 2012) were assessed using the DeLong method. Study outcomes included Disease Severity Index (DSI) and portosystemic shunt (SHUNT%).

[0307] Results: For the measurement of DSI, MM Vd The TRIO (v1.0) cholate test demonstrated similar ICCs but improved CVs compared to the MM cholate test method, as shown in Figure 10A and Table 13A. For the measurement of SHUNT%, the MM Vd The TRIO (v1.0) cholate test version demonstrated improved reproducibility based on both ICC and CV%. Vd The diagnostic performance based on AUROC for the TRIO (v1.0) cholate test version was comparable to that of MM in the majority of cases, and the SHUNT% was comparable to that of MM. Vd has been improved.

[0308] Conclusion: Next generation cholate test MM Vd and TRIO (v1.0) excludes samples for 5 minutes (MM Vd), reducing the total samples required to two (TRIO v1.0), and shortening the time from 90 to 60 minutes (TRIO v1.0). These improvements should enhance operator performance, resource utilization, and patient acceptance of the cholate test procedure, thereby enabling greater use of the cholate test to measure liver function and physiology.

[0309] Example 1B. Simplified Cholate Test: DuO Cholate Test for Prediction of Large Esophageal Varices Background: Endoscopy (EGD) is indicated in patients with cirrhosis to investigate large esophageal varices (LEV) requiring treatment. The prevalence of LEV on EGD in Child-Pugh (CP) A cirrhosis is approximately 10%. The cholate SHUNT test quantifies liver function and portosystemic shunting. An oral-only version (DuO cholate test) was developed to simplify test administration and reduce variability.

[0310] Objectives: The two aims of this study were to evaluate the diagnostic performance of the DuO cholate test in ruling out LEV in CP A cirrhosis and to compare the performance of the HepQuant DuO test with that of the cholate SHUNT test.

[0311] Methods: Subjects included 238 patients with CP A cirrhosis in the SHUNT-V study, including 52% MASLD / MASH, 25% HCV, and 16% alcoholic liver disease. Subjects included 64% obese, 87% overweight, and 54% diabetes.

[0312] SHUNT Test Implementation: 13C-cholate was infused intravenously, and d4-cholate was administered orally to subjects. Blood samples were collected for serum cholate at 0, 5, 20, 45, 60, and 90 minutes. Simplified cholate test versions included the cholate SHUNT V1.1 test and the DuO cholate test. The SHUNT V1.1 cholate test included all 13C-CA and d4-CA concentrations except for the 5-minute data point, which were calculated based on cubic spline and exponential fitting. Everson GT et al., Aliment. Pharmacol. Ther. 2007;26:401-410. The DuO cholate test included only d4-CA concentrations at 20 and 60 minutes, calculated based on a compartmental model. McRae MP et al., Transl. Res. 2023;252:53-63.

[0313] Study parameters: Study parameters were Disease Severity Index (DSI), portosystemic shunt (SHUNT%), hepatic reserve, and portal vein hepatic filtration rate (HFR P ) was included.

[0314] A DSI cutoff of <18.3 was prespecified based on the >95% sensitivity of LEV in the HALT-C trial QLFT adjunct study.

[0315] Statistical analyses included subgroup differences analyzed by ANOVA (continuous data) and chi-square (categorical data); DSI from lean and overweight controls plotted side-by-side with subjects with no esophageal varices, small esophageal varices, or large esophageal varices; diagnostic performance (AUROC, sensitivity, specificity, PPV, NPV) for ruling out LEV; AUROC for DuO; and SHUNT V1.1 for ruling out LEV compared by the DeLong method; and univariate logistic regression of DSI for the presence of LEV.

[0316] Results: Calculated laboratory values, clinical scores, and cholate test parameters from DuO in SHUNT-V CP A subjects for all subjects (n=238), subjects without varicose veins (n=135), subjects with small varicose veins (n=76), and subjects with large varicose veins (n=27) are shown in Table 13B.

[0317] As shown in Table 13B, simplified DuO cholate test parameters including DSI, SHUNT%, hepatic reserve, and HFRp were each sensitive in detecting the presence and size of varices.

[0318] [Table 10]

[0319] The DSI from lean and overweight controls was plotted alongside subjects with no esophageal varices, small esophageal varices, or large esophageal varices. As shown in Figure 10B, a monotonic stepwise increase in DSI with increasing risk for LEV was observed.

[0320] The DSI values ​​measured by the DuO cholate test in Child-Pugh A cirrhosis subjects and control subjects against the probability of LEV based on DSI are shown in Figure 10C. The DSI cutoff of 18.3 is shown as a vertical line. DSI from DuO demonstrated a significant association with the detection of LEV at endoscopic examination (p<0.001).

[0321] The diagnostic performance (95% CI) of DuO (DSI<18.3) in SHUNT-V CP A subjects (n=238) is shown in Table 13C.

[0322] Application of a DSI <18.3 cutoff from DuO for SHUNT-V subjects missed only 1 LEV case and prevented 84 EGDs compared with DSI from SHUNT V1.1.

[0323] [Table 11]

[0324] A pairwise comparison of the AUROC for DuO or DSI from SHUNT V1.1 in detecting LEV in SHUNT-V CPA subjects (n=238) is shown in Table 13D, which illustrates that DuO was equivalent to SHUNT V1.1 in detecting the presence of LEV.

[0325] [Table 12]

[0326] Conclusion: Simplified Liver Function and Physiology: Cholate DuO test parameters correlate with the presence and size of esophageal varices. Knowing the likelihood that a given DSI is associated with a particular risk of LEV is critical for clinical decision-making. DuO and SHUNT V1.1 were statistically equivalent in detecting LEV. DuO missed one case of LEV detected by SHUNT V1.1. Application of a DSI <18.3 avoided EGD in 35% of cases for DuO and 31% for SHUNT V1.1. DuO is easier to perform and less invasive, and therefore has the potential to be more widely accepted by healthcare providers performing the test and by patients undergoing the test.

[0327] Example 2. Reproducibility of simplified liver function tests In this example, the reproducibility of simplified liver function tests is described compared to that of SHUNT V1.0. This study was a retrospective analysis of data from two reproducibility studies: the HepQuant SHUNT study (236 tests completed in 94 subjects), the HepQuant Reproducibility Study (REPRO), and the Primary Sclerosing Cholangitis (PSC) study.

[0328] This example investigated the reproducibility of the DuO cholate test, an oral-only cholate tolerance test, and other simplified versions of the original SHUNT V1.0. The simplified tests, SHUNT V1.1, SHUNT V2.0, and DuO cholate test, reduced blood draw to four (SHUNT V1.1) or two samples (SHUNT V2.0 and DuO), made possible by a compartmental model (McRae et al., Transl Res. 2023;252:53-63), and, in the case of DuO, eliminated IV infusion. The intraindividual reproducibility of the simplified tests was measured.

[0329] subject The REPRO study included three groups of subjects: healthy individuals without liver disease, patients with NASH, and patients with HCV. The NASH group was diagnosed based on risk factors (obesity, diabetes, and metabolic syndrome), negative tests for other liver diseases, and fibrosis stage by liver biopsy or transient elastography. The HCV group was diagnosed based on a history of positive HCV via nucleic acid testing and METAVIR fibrosis stage determined by liver biopsy. Three separate HepQuant SHUNT tests were performed on three different days within a 30-day period.

[0330] Data from the PSC study (Everson GT, Helmke SM. Defining disease severity and measuring progression in primary sclerosing cholangitis (PSC): a comparison of the disease severity index (DSI) from the HepQuant SHUNT test with serum alkaline phosphatase (alk phos). Hepatology. 2018;68:1087A) were retrospectively analyzed. Reproducibility was assessed by performing two baseline SHUNT tests in 46 subjects representing the clinical spectrum of PSC.

[0331] The REPRO and PSC studies were conducted in accordance with the World Medical Association Code of Ethics (Declaration of Helsinki). Informed consent was obtained from all subjects. The REPRO study was approved by the Colorado Multi-Institutional Review Board and registered with ClinicalTrials.gov, NCT01579162. The PSC study was approved by the University of Colorado Denver Institutional Review Board, and cholate isotopes were studied under IND65121 (13C-CA) and IND65123 (d4-CA).

[0332] Reproducibility analysis Reproducibility of test parameters was assessed via intraclass correlation coefficient (ICC), coefficient of variation (CV), and minimal detectable difference (MDD). The ICC method was a single-rater / measure two-way mixed-effects model for absolute agreement between measurements (Koo et al., Guideline of Selecting and Reporting Intraclass Correlation Coefficients for Reliability Research. J Chiropr Med. 2016;15:155-63; McGraw et al., Forming inferences about some intraclass correlation coefficients. Psychol Methods. 1996;1:30-46), with a one-sided test for a lower acceptance limit (ICC > 0.7), previously defined as a reduction in precision of less than 5% (Burton et al., The within-individual reproducibility of the disease severity index from the HepQuant SHUNT test of liver function and physiology. Transl Res. 2021;233:5-15).

[0333] Acceptable reproducibility was determined by an ICC exceeding the lower tolerance limit of 0.7 (p<0.05). The MDD (Matheson GJ. We need to talk about reliability: making better use of test-retest studies for study design and interpretation. PeerJ. 2019;7:e6918), interpreted as the smallest detectable difference in the value of a test parameter before and after an experimental manipulation (e.g., treatment) that is greater than would be expected by chance, was calculated for each test parameter. Weir JP. Quantifying test-retest reliability using the intraclass correlation coefficient and the SEM. J Strength Cond Res. 2005;19:231-40. Baumgartner et al., Statistical evaluation of test-retest studies in PET brain imaging. EJNMMI Research. 2018;8:13.

[0334] Reproducibility was assessed for each study independently (three paired trials per subject for REPRO and two paired trials per subject for PSC) and combined for both studies. For the combined analysis, three pairs of two trials per subject were derived for the REPRO study data according to study visit (V): V1-V2, V1-V3, and V2-V3.

[0335] result Target characteristics REPRO Study. The REPRO study included 48 subjects divided into three groups: control (N = 16), NASH (N = 16), and HCV (N = 16). Subject characteristics (Table 15) and standard laboratory test results (Table 16) from the REPRO study (Burton et al., Transl Res. 2021;233:5-15) are reproduced here. The control group had a mean age of 32.9 ± 12.0 years, a balanced male-to-female ratio of 8:8, and a normal BMI of 23.0 ± 2.2. The NASH group had a mean age of 49.8 ± 11.6 years, a male-to-female ratio of 7:9 (M:F), and a high BMI of 32.5 ± 5.9. The HCV group had a mean age of 55.6 ± 6.7 years, a male-to-female ratio of 13:3, and a moderate BMI of 28.2 ± 4.1. The majority of subjects (40 of 48) were non-Hispanic white. Compared with controls, both the NASH and HCV groups had significantly higher levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), and glucose, and lower platelet counts. The NASH and HCV groups were classified based on fibrosis stage as either F0-F2 (early) or F3-F4 (advanced), with eight subjects in each stage. All F4 subjects had well-compensated liver disease without a history of clinical complications.

[0336] [Table 13]

[0337] PSC Study. Subject characteristics (Table 16) and standard laboratory test results (Table 15) from the PSC study were recorded. Of the 47 participants in the study, 43 were non-Hispanic white, 3 were African American, and 1 was Hispanic. The male to female ratio was 36:11, with a mean age of 48.8 ± 12.9 years, weight of 81.3 ± 14.9 kg, and blood pressure of 26.2 ± 3.9 kg / m. 2The BMI was 70% (33) of the participants had inflammatory bowel disease (IBD), and 76% (25) had ulcerative colitis. Of the participants, 36% (17) had a history of varices, ascites, or encephalopathy; 15 had varices, 2 had variceal bleeding, 5 had diuretic-responsive ascites, and 8 had treatment-responsive encephalopathy. Radiological reports indicated that 36% (17) had splenomegaly, and 28% (13) had a pleural effusion of 140,000 μL or more. -1 Patients had platelet counts below 0.05. Additionally, 19% (9 patients) had a history of jaundice, 38% (18 patients) had a history of bacterial cholangitis, and 72% (28 of 39 patients) of patients with endoscopic retrograde cholangiopancreatography (ERCP) reports had undergone stricture dilation, stent placement, or both. Treatment with ursodeoxycholic acid was administered to 66% (31 patients) of participants, and 40% (19 patients) were taking IBD medications.

[0338] [Table 14]

[0339] reproducibility REPRO Study. In the REPRO study (Table 17) (N=48 subjects), all study parameters were reproducible (ICC>0.7, p<0.05) for study versions SHUNT V1.1, SHUNT V2.0, and DuO. SHUNT% and RCA-20 did not meet the reproducibility criteria for SHUNT V1.0. STAT, portal vein HFR, and DSI were reproducible for study version STAT.

[0340] [Table 15]

[0341] The reproducibility of test parameters was assessed across fibrosis stages (N = 16 subjects for each fibrosis group and each subject category). Test parameters were calculated from the AUC of serum concentrations of cholate isotopes. AUC increased with increasing fibrosis stage and was lowest in healthy individuals and patients with low fibrosis stages. Low AUC was associated with relatively large discrepancies. As a result, ICCs were higher for each test parameter and test version with increasing fibrosis stage (data not shown) and in diseased subjects compared with controls (data not shown). DSI, hepatic reserve, and risk ACE met reproducibility criteria across F0-F2 fibrosis for SHUNT V1.0 and SHUNT V1.1, and across F3-F4 for all test versions (p < 0.001). Point estimates of ICC were not statistically significant (p<0.05) in individual cases, but generally exceeded the reproducibility cutoff of 0.70 across all test parameters and test versions.

[0342] PSC Study. In the PSC study (Table 18) (N=46 subjects), all test parameters met the criteria for reproducibility (ICC>0.7, p<0.05) across all test versions except for whole-body HFR and RCA-20 (p<0.001).

[0343] [Table 16]

[0344] Pooling. Overall, HepQuant study parameters were highly reproducible across study versions (ICC values ​​of approximately 0.94 for DSI, approximately 0.89 for SHUNT%, and approximately 0.95 for hepatic reserve). For study parameters emphasizing systemic clearance (systemic HFR, SHUNT%, RCA-20), CV and MDD decreased in the following order from highest to lowest variability: SHUNT V1.0, SHUNT V2.0, SHUNT V1.1, DuO. In both pooled studies, DuO had a substantially lower MDD for SHUNT% (3.56) than SHUNT V1.0 (7.80) (Table 19). For study parameters emphasizing portal vein clearance (portal HFR, DSI, hepatic reserve, risk ACE), CV and MDD increased slightly or remained the same for SHUNT V2.0 and DuO. Regarding hepatic reserve, which had the lowest CV of all test parameters at approximately 2-3%, SHUNT V1.0 and SHUNT V1.1 were similar in terms of MDD (2.47 and 2.58, respectively), while SHUNT V2.0 and DuO were slightly higher (3.24 and 3.28, respectively). Regarding DSI, SHUNT V1.1 had the lowest MDD (1.40), followed by SHUNT V1.0, SHUNT V2.0, and DuO (1.60, 1.67, and 1.73, respectively).

[0345] [Table 17]

[0346] The simplified method was found to be highly reproducible across study parameters, with intraclass correlation coefficients of less than 0.93 for the primary study parameters (DSI and hepatic reserve). SHUNT V2.0 and DuO improved reproducibility in the measurement of portosystemic shunt (SHUNT%).

[0347] Example 3. Equivalence of simplified liver function tests to the cholate SHUNT test The equivalence of the double-sample oral cholate challenge test (DuO) to the cholate SHUNT test and other simplified versions of the cholate SHUNT test was investigated.

[0348] Current non-invasive liver tests primarily function as surrogates for fibrosis and lack the ability to directly measure liver function. The cholate SHUNT liver function test directly addresses this issue by utilizing the liver uptake of stable cholate isotopes to measure liver function and physiology. Because the HepQuant SHUNT test (V1.0 / 1.1) is complex to perform, simplified test versions SHUNT V2.0 (oral and IV dosing, but only two blood samples at 20 and 60 minutes) and DuO (oral dosing only, only two blood samples at 20 and 60 minutes) were developed as described herein. The excellent reproducibility and reliability of a single test within a given individual are also described herein.

[0349] In this example, the equivalence of these simplified tests to the original cholate SHUNT liver function test was investigated. Data from three studies involving 930 cholate SHUNT tests in 372 subjects were retrospectively analyzed by each method.

[0350] The cholate SHUNT liver function test is a unique testing platform suitable for addressing the spectrum of liver disease etiology and severity through evaluation of both hepatocellular function and portal circulation. (Everson et al., Portal-systemic shunting in patients with fibrosis or cirrhosis due to chronic hepatitis C: the minimal model for measuring cholate clearances and shunt. Aliment Pharmacol Ther. 2007;26:401-10.) (Everson et al., The spectrum of hepatic functional impairment in compensated chronic hepatitis C: results from the Hepatitis C Anti-viral Long-term Treatment against Cirrhosis Trial. Aliment Pharmacol Ther. 2008;27:798-809.) Despite the strong promise of the cholate SHUNT liver function test, its implementation involves administering both oral and intravenous (IV) cholate doses and collecting five peripheral venous blood samples over a 90-minute period. This process introduces possible sources of error, such as difficulties with IV access, risk of extravasation, timing errors during sample collection, and variability in test administration skill.

[0351] A compartmental model for the dual cholate clearance assay was previously developed to enhance its reproducibility and interpretation of hepatic extraction and portosystemic shunting. McRae et al., Compartmental model describing the physiological basis for the HepQuant SHUNT test. Transl Res. 2023;252:53-63.

[0352] Using a compartmental modeling approach, simplified versions of the cholate SHUNT test, SHUNT V2.0 and DuO, were developed, which require fewer blood draws, reduce test time, simplify test administration, and enhance the reproducibility of test parameters. The cholate SHUNT V1.1 liver function test, which eliminates the need for a 5-minute sample, was found to be more reproducible than SHUNT V1.0 and was therefore designated as the reference method for equivalence assessment herein. This example investigates whether the simplified DuO and SHUNT V2.0 tests are equivalent to the reference (SHUNT V1.1) method.

[0353] method This study was a retrospective analysis of data collected from three different studies of the HepQuant SHUNT trial: the HepQuant Reproducibility Study (REPRO) (Burton et al., The within-individual reproducibility of the disease severity index from the HepQuant SHUNT test of liver function and physiology. Transl Res. 2021;233:5-15), the Primary Sclerosing Cholangitis (PSC) Study (Everson and Helmke. Defining disease severity and measuring progression in primary sclerosing cholangitis (PSC): a comparison of the disease severity index (DSI) from the HepQuant SHUNT test with serum alkaline phosphatase (alk phos). Hepatology. 2018;68:1087A), and the Hepatitis C Antiviral Long-Term Treatment Trial for Cirrhosis (HALT-C) Quantitative Liver Function Testing (QLFT) Substudy (Everson et al., Quantitative liver function tests improve the prediction of clinical outcomes in chronic liver disease). hepatitis C: Results from the hepatitis C antiviral long-term treatment against cirrhosis trial.Hepatology.2012;55:1019~29).

[0354] subject The REPRO study included 48 subjects from three groups (16 control, 16 NASH, and 16 HCV) who underwent three separate SHUNT tests on three different days within 30 days, representing a total of 144 SHUNT tests conducted in the study. Healthy controls had no history of liver disease and normal standard blood tests. NASH was diagnosed based on risk factors, the absence of evidence of other liver disease, and fibrosis staging by liver biopsy or transient elastography. HCV was diagnosed based on confirmed HCV history by nucleic acid testing and METAVIR fibrosis staging by liver biopsy.

[0355] The PSC study included 47 patients with PSC recruited from hepatology clinics, representing the clinical spectrum of PSC. All 47 subjects underwent a baseline SHUNT test, 46 underwent a second baseline test to assess reproducibility, and 40 underwent a follow-up test one year later, representing a total of 133 SHUNT tests performed in the study.

[0356] Details of the HALT-C QLFT study have been published previously (Everson et al., Hepatology. 2012;55:1019-29). Cholate SHUNT studies were performed in 277 subjects at baseline, 212 at 2 years, and 164 at 4 years. A total of 653 SHUNT studies were performed in the HALT-C study.

[0357] Cholate Liver Function Test Version The cholate liver function test versions are summarized below. The cholate SHUNT V1.0 liver function test involves the simultaneous administration of IV 13C-CA and oral d4-CA and six peripheral venous blood samples over 90 minutes. Serum cholate concentrations were measured by LC / MS, and noncompartmental analysis was performed by fitting IV and oral clearance curves to the measured data to determine the IV and oral areas under the curve (AUC IV , AUC Oral) (Everson et al., Portal-systemic shunting in patients with fibrosis or cirrhosis due to chronic hepatitis C: the minimal model for measuring cholate clearances and shunt. Aliment Pharmacol Ther. 2007;26:401-10).

[0358] The cholate SHUNT V1.1 liver function test excludes the 5-minute sample from the calculation. Instead, C-CA is estimated from the total blood volume calculation (Lemmens et al., Estimating blood volume in obese and morbidly obese patients. Obes Surg. 2006;16:773-6), and the 5-minute d4-CA is estimated by 15% of the 20-minute concentration. This modification has been shown to significantly reduce variability in the measurement of test parameters that depend on systemic clearance. For this reason, SHUNT V1.1 is designated as the reference method in this example.

[0359] The Cholate SHUNT V2.0 Liver Function Test further simplifies the test by shortening the test window and requiring only two blood draws at 20 and 60 minutes. The Cholate SHUNT V2.0 uses a compartmental model to measure portal venous cholate clearance (McRae et al., Transl Res. 2023;252:53-63) and non-compartmental exponential fitting to measure systemic cholate clearance. The DuO cholate liver function test is an oral-only test that involves a single oral dose at 0 minutes and two blood samples taken at 20 minutes and 60 minutes. IV clearance is induced rather than measured, and the induced IV concentrations are then used in the same non-compartmental analysis as SHUNT V2.0.

[0360] The cholate STAT liver function test is the simplest test, involving a single oral cholate dose and a single blood sample over 60 minutes. STAT test values ​​can be reported as either a STAT score (e.g., d4-CA concentration adjusted to 75 kg body weight), a STAT-estimated portal vein HFR, or a STAT-estimated DSI.

[0361] Test parameters Various cholate liver function tests return a range of test parameters that have previously been associated with liver function and disease / health. This study evaluated the following cholate test parameters for their equivalence to a reference method:

[0362] Portal hepatic filtration rate (HFR) P ) is the portal venous clearance (unit: mL min ) adjusted for body weight -1 kg -1 ) and calculated by Equation 7B:

number

[0363] Whole body hepatic filtration rate (HFR) S ) is the measured systemic clearance (in mL min ) adjusted for body weight. -1 kg -1 ) and calculated by Equation 12:

number

[0364] The Disease Severity Index (DSI) is a score ranging from 0 to 50 that indicates overall liver function, including both portal and systemic HFR. The DSI is related to the fibrosis stage and clinical stage of cirrhosis

[24] and was calculated by equation 14:

number

[0365] where A is the magnification and HFR P,max and HFR S,max is the upper limit of clearance relative to healthy controls.

[0366] SHUNT%, or shunt fraction, is the estimated absolute bioavailability of an oral d4-CA dose in the systemic circulation as estimated by Equation 9B. SHUNT% is a direct measure of first-pass hepatic extraction of cholate, which is affected by portal blood flow and the portosystemic shunt.

[0367]

number

[0368] Hepatic reserve (HR) represents an individual's overall liver health compared to lean healthy controls, with a value ranging from 100 (healthy) to 0 (severely impaired). HR was calculated according to Equation 15:

[0369]

number

[0370] where A is the magnification and HFR P,lean and HFR S,lean is the mean HFR-1 standard deviation from a population of lean controls.

[0371] statistical analysis Cholate liver function testing can potentially be affected by inconsistencies in test compound administration and sample collection times, day-to-day variations in gastrointestinal or hepatic physiology, and variability in sample preparation and laboratory analysis. Upper and lower equivalence boundaries were identified based on the desired minimum effect size defined by the 95% confidence interval for intraindividual variability from the REPRO study, as shown in Table 20.

[0372] [Table 18]

[0373] To assess agreement, correlation plots compared the analytical method with a reference method (SHUNT V1.1) for determination of test parameters using Deming regression fitting to assess systematic differences. Cornbleet et al., Incorrect least-squares regression coefficients in method-comparison analysis. Clin Chem. 1979;25:432-8.

[0374] Bland-Altman plots compared test parameters calculated by each analysis method and the reference method with respect to bias and 95% confidence intervals (CI). Bland JM, Altman DG. Measuring agreement in method comparison studies. Stat Methods Med Res. 1999;8:135-60.

[0375] The primary performance measure was the proportion of trials / subjects in which the difference between analysis methods was smaller than any clinically meaningful difference. Analyses were completed for each study separately and for all datasets combined.

[0376] The FDA has also provided guidelines on preferred methods for demonstrating test equivalence—the double-tailed one-tailed t-test (TOST) (Lakens D. Equivalence Tests: A Practical Primer for t Tests, Correlations, and Meta-Analyses. Social Psychological and Personality Science. 2017;8:355-62; Rogers et al., Using significance tests to evaluate equivalence between two experimental groups. Psychol Bull. 1993;113:553-65) and bioequivalence procedures (U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Statistical Approaches to Establishing Bioequivalence—Guidance for Industry. Rev. 1 ed. 2022).

[0377] In the TOST procedure, upper and lower equivalence boundaries were identified based on the desired minimum effect size (Table 20). Two composite null hypotheses were tested. If both one-sided tests are statistically rejected, the observed effect falls within the equivalence boundaries and is statistically smaller than either effect that is considered valuable and practically equivalent. To assess bioequivalence, mean ratios and 90% confidence intervals for the Next Generation Study and V1.0 were calculated for each study separately and across all studies. The mean ratios and 90% CIs were compared to the 80% and 120% bioequivalence limits for the parameters in their original scale (i.e., not log-transformed).

[0378] Target characteristics REPRO Study. The REPRO study included 48 participants divided into three groups: control (N = 16), NASH (N = 16), and HCV (N = 16). The control group had a mean age of 33 ± 12 years, an equal male-to-female distribution (8:8), and a normal BMI of 23 ± 2. The NASH group had a mean age of 50 ± 12 years, a male-to-female ratio of 7:9, and an elevated BMI of 33 ± 6. The HCV group had a mean age of 56 ± 7 years, a male-to-female ratio of 13:3, and a moderate BMI of 28 ± 4. The majority of participants (83%) were non-Hispanic white. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), and glucose were elevated in NASH and HCV compared with controls. Platelets were significantly lower in NASH and HCV. Both the NASH and HCV groups had 8 patients with F0-F2 (early) fibrosis and 8 patients with F3-F4 (advanced) fibrosis.

[0379] PSC Study. Of the 47 subjects in the PSC study, 92% were non-Hispanic white, 6% were African American, and 2% identified as Hispanic. The majority of subjects were male (36:11 M:F), with a mean age of 49 ± 13 years, weight of 81 ± 15 kg, and a mean blood pressure of 26 ± 4 kg / m. 2 Seventy percent of the subjects had inflammatory bowel disease, 53% had ulcerative colitis, 36% had a history of varices, ascites, or encephalopathy, 36% had splenomegaly, and 28% had low platelet counts (<140,000 μL). -1 ), 19% had a history of jaundice, and 38% had a history of bacterial cholangitis. Of subjects with ERCP reports, 72 percent (28 of 39) had undergone stricture dilatation, stent placement, or both. 66 percent of subjects had been treated with UDCA, and 40% were taking medication for inflammatory bowel disease.

[0380] HALT-C QLFT Study. Of 285 chronic HCV patients enrolled in the HALT-C trial QLFT substudy, 60% had Ishak fibrosis stages 2-4 (fibrosis) and 40% had stages 5 or 6 (cirrhosis). No subjects experienced clinical decompensation. The majority of subjects were male (76%), with a mean age of 49.9 ± 7.2 years and a mean BMI of 29.4 ± 4.8. Ninety-two percent had a 6.4 (± 0.56) log IU / mL vasodilator. 10 The patients had HCV genotype 1 and a mean (±SD) HCV-RNA of 169,000 (±69,000) μL. -1 It was.

[0381] Equivalence to SHUNT V1.1 The equivalence of SHUNT V2.0, DuO, and STAT to SHUNT V1.1 is summarized in Table 21, which shows the percentage of studies falling within the equivalence bands for each study individually and across all studies. Equivalence in DSI between the simplified test versions and SHUNT V1.1 was further assessed by TOST and bioequivalence methods (Figures 14A-D).

[0382] [Table 19]

[0383] SHUNT V2.0. SHUNT V2.0 was equivalent to the reference method for all test parameters, with the percentage of tests within the equivalence boundaries ranging from 97% to 100%. The DSI measured by SHUNT V2.0 was consistent with a Deming regression line (R ) approaching identity, with low bias (0.21 DSI units) and all but seven tests falling within the equivalence boundaries. 2SHUNT V2.0 had excellent agreement with the reference method for DSI, with an R of 0.96 (R = 0.98, slope = 0.99, intercept = -0.05) (Figures 15A-B). DSI from SHUNT V2.0 was also statistically equivalent, not different, and bioequivalent to DSI measured from SHUNT V1.1, both individually and across all studies combined (Figure 14B). SHUNT% measured by SHUNT V2.0 also had an average bias of 1.3% and an R of 0.96. 2 There was excellent agreement with the reference method, with (data not shown).

[0384] DuO. DuO was comparable to the reference method with respect to DSI (97%), portal vein HFR (99%), and hepatic functional reserve (95%), but not with respect to SHUNT% (85%) and systemic HFR (82.5%). DSI from DuO was plotted along a nearly identical Deming regression line (R 2 = 0.95, slope = 1.01, intercept = -0.16), demonstrating excellent agreement with the reference method (Figures 16A-B). DSI from DuO was found to be statistically equivalent, not different, and bioequivalent to SHUNT V1.1, with a slightly larger 90% confidence interval than that for SHUNT V2.0 (Figure 14D). SHUNT% estimated by DuO had modest agreement with the reference method (data not shown) (R 2 = 0.86, slope = 0.87, intercept = 0.004). DuO demonstrated excellent agreement with respect to hepatic reserve (Figures 18A-B) (R 2 = 0.97, slope = 1.01, intercept = 0.01). Additionally, hepatic functional reserve from DuO was found to be statistically equivalent, not different, and bioequivalent to SHUNT V1.1 by TOST and bioequivalence methods (Figures 19A-D).

[0385] STAT. For the STAT test, portal vein HFR (98%) was comparable to the reference method, but DSI (85%) was not. The DSI estimated by the STAT test (Figures 17A-17B) had a relatively low bias (-0.93 DSI units), but its overall agreement with the reference method was modest in terms of correlation (R 2 =0.89, slope=0.92, intercept=2.52).

[0386] Equivalence to SHUNT V1.0 A secondary analysis was performed using SHUNT V1.0 as the reference method. The results are summarized in Table 22.

[0387] [Table 20]

[0388] SHUNT V1.1 was comparable to SHUNT V1.0 for DSI, SHUNT%, hepatic reserve, portal HFR, and systemic HFR, with >98% of values ​​within the equivalence boundaries. DSI measured by SHUNT V1.1 was consistent with low bias (0.21 DSI units) and a Deming regression line (Figures 20A-20B) that approached identity (R 2 = 0.99, slope = 1.00, intercept = -0.15). Compared to SHUNT V1.1 as the reference method, the simplified test version had a demonstrated level of identity with SHUNT V1.0 as the reference method.

[0389] This example used several methods to confirm the equivalence of the DuO cholate liver function test and DSI from the SHUNT V2.0 study to DSI from the more complex SHUNT study (V1.0 / 1.1). Table 23 summarizes the key findings from this study as well as those from the reproducibility analysis described above.

[0390] Table 23 shows a summary of the equivalence studies for the simplified methods (SHUNT V2.0 and DuO) compared to SHUNT V1.1 and the reproducibility by whether the criteria were met (Y = yes, N = no). For equivalence, the criteria were ≥ 95% of the studies within the equivalence boundary, double-tailed one-tailed t-test (TOST), and bioequivalence (BE). For reproducibility, the criteria were an intraclass correlation coefficient (ICC) > 0.7.

[0391] [Table 21]

[0392] The DSI from SHUNT V1.0 has been associated with numerous important laboratory tests, clinical models, clinical complications, and clinical outcomes. By demonstrating equivalence and high intra-individual reproducibility to the DSI from SHUNT (V1.0 / 1.1), the DSI from DuO or SHUNT V2.0 is likely to demonstrate the same association with clinical endpoints.

[0393] Simplified versions of the two-sample oral dose cholate tolerance test (DuO) and the cholate SHUNT liver function test (SHUNT V2.0) have been demonstrated to produce results equivalent to the original cholate SHUNT test in a shorter time and requiring fewer blood samples. The cholate SHUNT V2.0 and DuO liver function test methods were found to be equivalent to the original cholate SHUNT test with respect to Disease Severity Index (DSI), with over 99% and over 96% of tests, respectively, falling within the equivalence boundaries. Both DuO and SHUNT V2.0 met the additional equivalence criteria set forth in FDA guideline documents: double-sided, one-tailed t-test and bioequivalence. The DuO and cholate SHUNT V2.0 are easier to perform and less invasive than the original SHUNT test, potentially making them more widely accepted by healthcare providers and patients.

[0394] Example 4. Prediction of clinical outcome in primary sclerosing cholangitis In this example, liver function and portosystemic shunt were quantified in primary sclerosing cholangitis (PSC) using the cholate SHUNT V1.1 test and the DuO cholate test and cholate SHUNT V2.0, which are simplified tests based on a compartmental model as described herein. The test results were compared with standard laboratory tests and clinical models in predicting clinical outcomes.

[0395] Objectives: The two objectives of this study were to determine whether the simplified cholate tests DuO and SHUNT V2.0 can predict clinical outcome in PSC and to measure the reproducibility of the tests.

[0396] Methods: The study included 47 patients spanning the clinical spectrum of PSC: 46 patients were retested at baseline for reproducibility, and 40 patients were followed prospectively for clinical outcomes and retested after 1 year.

[0397] The cholate SHUNT test administration included 13C-cholate infused via IV and d4-cholate administered orally to subjects. Blood was collected for serum cholate at 0, 5, 20, 45, 60, and 90 minutes. The simplified cholate test versions included the Cholate SHUNT V1.1, Cholate SHUNT V2.0, and DuO Cholate tests. The SHUNT V1.1 test included all 13C-CA and d4-CA concentrations except for the 5-minute data point. The Cholate SHUNT V2.0 test included 13C-CA and d4-CA concentrations at 20 and 60 minutes. The DuO Cholate test included only d4-CA concentrations at 20 and 60 minutes.

[0398] Study parameters included the Liver Disease Severity Index (DSI), portosystemic shunt (SHUNT%), hepatic functional reserve (HR), and portal hepatic filtration rate (HFRP). Statistical analysis included three subgroups of PSC progressors characterized by the degree of liver damage with age: slow (n = 28), intermediate (n = 16), and rapid (n = 3).

[0399] Reproducibility was determined by intraclass correlation coefficient (ICC), minimal detectable difference (MDD), and coefficient of variation (%CV); an ICC > 0.7 was considered acceptable.

[0400] AUROCs were compared across test versions for prediction of clinical outcomes (new clinical decompensation, liver-related death, liver transplant) using the DeLong method.

[0401] Logistic regression of baseline SHUNT% on portal hypertension and varicose veins was calculated.

[0402] Results: Cholate test parameters from DuO, including Liver Disease Severity Index (DSI), portosystemic shunt (SHUNT%), hepatic functional reserve (HR), and hepatic portal vein filtration rate (HFRP), laboratory and clinical test scores for PSC slow progressors (n=28), intermediate and rapid progressors (n=19), and group t-test p-values ​​are shown in Table 24.

[0403] [Table 22]

[0404] Graphs of the PSC progressor group showing subject age versus DSI values ​​based on DSI from DuO are shown in Figure 21A. Graphs of the PSC progressor group showing subject age versus SHUNT% values ​​based on SHUNT% calculated from DuO are shown in Figure 21B.

[0405] PSC rapid progressors are characterized by a high SHUNT% at a relatively young age. As shown in Table 24 and Figure 21B, intermediate / rapid progressors were more likely to have poor DuO cholate test parameters, poor laboratory tests, and experience poor clinical outcomes compared with slow progressors.

[0406] Predictions of clinical outcomes and reproducibility for DSI and SHUNT% calculated by the SHUNT V1.1, SHUNT V2.0, and DuO cholate tests are shown in Table 25.

[0407] [Table 23]

[0408] As shown in Table 25, DSI and SHUNT% had excellent intra-individual reproducibility and were the strongest predictors of new clinical decompensation, liver-related death, or liver transplant (n=13), with no significant differences between test versions.

[0409] SHUNT% is related to the baseline characteristics of portal hypertension (varices, splenomegaly, platelets <140,000) as shown in Figure 22A, and varices as shown in Figure 22B.

[0410] Conclusions: Cholate test parameters of liver function and physiology correlate with laboratory and clinical evidence of PSC disease severity, identify progressor groups, and predict risk for clinical outcomes. There were no significant differences between test versions with respect to prediction of clinical outcome and reproducibility. The DuO and SHUNT V2.0 cholate tests are easier to perform and less invasive, and therefore have the potential to be more widely accepted by healthcare providers who perform the tests and by patients who receive them.

[0411] Example 5. The simplified cholate test measures risk reduction for clinical events in subjects with compensated NASH cirrhosis treated with resmetirom Nonalcoholic steatohepatitis (NASH) is a progressive liver disease for which there is no approved treatment. Generally, approximately 25% of patients with NAFLD have NASH, which is defined as the presence of liver fat content (steatosis) greater than 5% combined with liver cell injury (balloon degeneration) and inflammation.

[0412] MAESTRO-NAFLD-1 (NCT04197479) was a 52-week Phase 3 clinical trial to evaluate the safety and tolerability of resmetirom, a thyroid hormone receptor beta agonist being investigated for the treatment of NASH (Harrison et al., Lancet 2019, 394:2012-24). MAESTRO-NAFLD-1 included an open-label, active resmetirom treatment arm in patients with well-compensated (Child-Pugh A [CP-A]) NASH cirrhosis (Figure 23).

[0413] The cholate SHUNT test quantifies liver function and physiology. Simplified versions (e.g., SHUNT V2.0 and DuO cholate test) require fewer blood samples and shorter test times.

[0414] The objective of this study was to determine whether the simplified cholate test, SHUNT V2.0, and the DuO cholate test could detect treatment effects in MAESTRO-NAFLD-1.

[0415] Methods: Subjects with compensated NASH cirrhosis (n=34) underwent baseline testing and subsequent retesting at weeks 28 and 48. Eligibility included at least three metabolic risk factors and liver biopsy-confirmed NASH cirrhosis or accepted criteria.

[0416] The cholate SHUNT study involved intravenous 13C-CA and oral d4-CA administration. Blood was collected at 0, 5, 20, 45, 60, and 90 minutes for serum cholate concentrations. AUC was calculated using a compartmental model (McRae et al., 2023 Translational Res. 252:53-63). For SHUNT V2.0: IV and oral data at 20 and 60 minutes were used. For DuO: Only oral data at 20 and 60 minutes were used.

[0417] The risk ACE was calculated for each subject from the Disease Severity Index (DSI) at baseline and at weeks 28 and 48. A Poisson model (risk ACE) estimated the annualized clinical event rate based on 220 subjects with 52 clinical events from the HALT-C trial. The result is the clinical event rate (clinical events per person-year).

[0418] Model A: Association with baseline DSI (indicated by dsi0): Y=β0+β1dsi0

[0419] Model D: Association between baseline DSI and change in DSI (indicated by dsiΔ): Y=β0+β1dsi0+β2dsi Δ

[0420] The difference from baseline is represented by the difference between Risk ACE Model A and Model D (Table 26).

[0421] [Table 24]

[0422] The cholate test versions DuO and SHUNT V2.0 significantly simplified test administration, reducing blood samples by two-thirds and test time by one-third, as shown in Figures 24A and 24B, which show intravenous and oral cholate clearance curves. Only cholate serum values ​​at 20 and 60 minutes post-dose were required.

[0423] The risk ACE calculated by the DuO cholate test as a change from baseline on resmetirom at 28 and 48 weeks is shown in Figures 25A and 25B, respectively.

[0424] The risk ACE calculated by the cholate SHUNT V2.0 study as a change from baseline with resmetirom at 28 and 48 weeks is shown in Figures 26A and 26B, respectively.

[0425] The risk ACE calculated by SHUNT V2.0 and Duo Cholate study versions as changes from baseline at weeks 28 and 48 are shown in Table 27.

[0426] [Table 25]

[0427] As shown in Table 27, risk ACE from DuO decreased with resmetirom treatment in 21 of 23 subjects, with a significant mean reduction at 28 weeks. At 48 weeks, risk ACE decreased in 19 of 23 subjects (-0.0355, p=0.1222). SHUNT V2.0 demonstrated similar reductions in risk ACE at 28 weeks (V2.0: -0.0170, p=0.1145) and 48 weeks (V2.0: -0.0325, p=0.1605).

[0428] Conclusions: The simplified cholate test measured a reduction in estimated clinical event rates after 28 weeks of resmetirom. The DuO cholate test provides a sensitive and interpretable measure of risk for all clinical events for monitoring patients. DuO and SHUNT V2.0 are easier to administer and less invasive, and therefore have the potential to be more widely accepted by healthcare providers administering the test and by patients undergoing the test.

[0429] Terms Clause 1. obtaining blood or serum sample concentration data of an orally administered identifiable cholate compound taken from a subject at two time points after oral administration; simulating a complete oral clearance curve using a compartmental model of oral cholate clearance, the compartmental model including body mass index (BMI), body weight (BW), and optionally hematocrit (Hct) input values ​​for the subject; and Calculating the area including the trapezoidal numerical integration to obtain the AUCoral measuring the area under the curve (AUCoral) of the blood or serum concentration of orally administered distinguishable cholate compounds in a subject; and calculating one or more indices of liver disease in a subject using AUCoral, wherein the one or more indices are related to liver function in the subject; 1. A method for assessing liver function in a subject having or suspected of having or suffering from liver disease, comprising:

[0430] Clause 2. Obtaining concentration data of orally administered distinguishable cholate compounds at two time points, receiving first and second blood or serum samples taken from the subject at first and second time points after a single oral dose of a first distinguishable cholate compound; and analyzing the sample to obtain oral concentration data at the first and second time points, optionally The blood or serum sample is collected within about 180 minutes, 120 minutes, 90 minutes, or about 75 minutes after oral administration. The method of clause 1, including

[0431] Clause 3. The method of Clause 2, wherein the first and second blood or serum samples are collected from the subject at least about 5 minutes to about 75 minutes, 10 minutes to 70 minutes, 20 minutes to 60 minutes, 25 minutes to 55 minutes, 30 minutes to 50 minutes, 35 minutes to 45 minutes, or about 40 minutes apart.

[0432] Clause 4. The method of claim 2 or 3, wherein the first and second blood or serum samples are taken from the subject about 20 minutes and about 60 minutes, respectively, after oral administration.

[0433] Clause 5. Obtaining concentration data of orally administered distinguishable cholate compounds at two time points, receiving a single blood or serum sample taken from the subject after administering to the subject a first oral dose of a first distinguishable cholate compound and a second oral dose of a second distinguishable cholate compound; and analyzing a single sample to obtain oral concentration data for a first distinguishable cholate compound and a second distinguishable cholate compound at two time points, optionally the single blood or serum sample is obtained from the subject within about 180 minutes, 120 minutes, 90 minutes, or about 75 minutes after administration of the first oral dose; The method of clause 1, including

[0434] Clause 6. The method of Clause 5, wherein the first and second oral doses are administered to the subject at least about 5 minutes to about 75 minutes, 10 minutes to 70 minutes, 20 minutes to 60 minutes, 25 minutes to 55 minutes, 30 minutes to 50 minutes, 35 minutes to 45 minutes, or about 40 minutes apart.

[0435] Clause 7. The method of clause 5 or 6, wherein the single sample is collected from the subject simultaneously about 20 minutes after the second oral dose and about 60 minutes after the first oral dose.

[0436] Clause 8. Estimating the area under the curve (AUCiv) of the blood or serum concentration of an intravenously administered distinguishable cholate compound; and calculating one or more indices of liver disease in the subject using the AUCoral and AUCiv values; 8. The method of any one of clauses 1 to 7, further comprising:

[0437] Clause 9. The step of estimating AUCiv includes a linear regression model, and optionally, the linear regression model includes Equation 11A: AUC IV =β0+β BW BW+β PO,20 C PO,20 +β PO,60 C PO,60 +β HFR,P HFR P Equation 11A where β0 is the intercept coefficient, optionally the intercept coefficient is 161.972; B BW is the weight coefficient, optionally the weight coefficient is 0.6459; β PO,20 is the orally administered discriminant cholate concentration coefficient at the first time point, and optionally β PO,20 is 16.9249; C PO,20 is the orally administered discernible cholate concentration at the first time point; β PO,60 is the orally administered discriminant cholate concentration coefficient at the second time point, and optionally β PO,60 is 89.2405; β PO,60 is the orally administered discernible cholate concentration at the second time point; and β HFR,P is the portal vein HFR coefficient, and optionally the portal vein HFR coefficient is -0.4755; Clause 8 Method.

[0438] Clause 10. The step of estimating AUCiv, obtaining blood or serum sample concentration data of the intravenously administered third identifiable cholate compound in one of the samples taken from the subject; and exponentially fitting the intravenous concentration data to a systemic cholate clearance curve including rapid, moderate, and slow phases of clearance over a period of at least about 180 minutes following iv administration of the intravenous dose. the methods of clause 8, including:

[0439] Clause 11. The method of clause 10, wherein the third identifiable cholate is administered intravenously simultaneously with the first oral dose or at least about 5 minutes to about 75 minutes, 10 minutes to 70 minutes, 20 minutes to 60 minutes, 25 minutes to 55 minutes, 30 minutes to 50 minutes, 35 minutes to 45 minutes, or about 40 minutes after the first oral dose.

[0440] Clause 12. The method of clause 10 or 11, wherein the blood or serum sample collected at a single time point is collected within about 90 minutes or less, 75 minutes or less, 60 minutes or less, 45 minutes or less, 30 minutes or less, or within about 20 minutes after intravenous administration.

[0441] Clause 13. The fit for the rapid phase (Y0) of the systemic cholate clearance curve is calculated according to equation 20:

number

number

number

number

[0442] Clause 14. The fit for the rapid phase (Y1) of the systemic cholate clearance curve is calculated according to equation 21:

number

[0443] Clause 15. The fit for the slow phase (Y2) of the systemic cholate clearance curve is calculated according to equation 22:

number

[0444] Clause 16. The area under each of the three exponential curve fits is calculated by trapezoidal numerical integration, AUC IV Any one of the methods in clauses 10 to 15, which are summed to estimate the

[0445] Article 17. A compartmental model of oral clearance is Compartment volumes of multiple compartments in a subject; and Flow parameters between multiple compartments in a subject 17. The method of any one of clauses 1 to 16, comprising the step of estimating

[0446] Clause 18. The method of clause 17, wherein the compartmental model further comprises estimating cholate binding and dose administration in the subject.

[0447] Clause 19. The method of clause 17 or 18, wherein the multiple compartments in the subject include systemic, portal, and hepatic compartments.

[0448] Clause 20. The step of estimating compartment volumes of a plurality of compartments comprises: The total body compartment volume (V S ), portal vein compartment volume (V P ), and liver compartment volume (V L ) 20. The method of clause 18 or 19, optionally wherein the volume of each compartment is in liters (L).

[0449] Article 21. Total Body Compartment Volume (V S ) is estimated according to equation 4A: V S =TBV·(1-Hct) Equation 4A During the ceremony, TBV is the total blood volume in the subject according to equation 4:

number

[0450] Article 22. Portal venous compartment volume (V P ) is estimated according to equation 4B: V P =0.25 V S Equation 4B.

[0451] Article 23. Liver compartment volume (V L ) is estimated according to equation 5A: V L =(0.275 22.46 BW d L f plasma ) / 1000 Equation 5A In the formula, d L is 1.06 g mL -1 is the hypothetical liver tissue density of Any one of the methods set forth in Articles 20 to 22.

[0452] Clause 24A. The process of estimating flow parameters between multiple compartments includes a system of first-order ordinary differential equations 1 to 3;

number

number

number

[0453] Clause 24B. The process of estimating flow parameters between a plurality of compartments includes a system of first-order ordinary differential equations 1B, 2B, and 3:

number

number

number

[0454] where V is the total body (V S ), portal vein (V P ), and liver (V L ) is the volume of each of the compartments, and C is the total body volume (C S ), portal vein (C P ), and liver (C L) the concentration of orally administered distinguishable cholate compounds in each of the compartments, q is the flow rate between the compartments, and Cl H is the hepatic clearance and D PO,rate is the rate at which orally administered identifiable cholate compounds enter the portal compartment, Any one of the methods set forth in Articles 17 to 23.

[0455] Clause 25. The step of estimating comprises estimating the total hepatic inflow (Q L ), splanchnic arterial circulation (q SP ), hepatic portal vein inflow to the liver (q PL ), total hepatic venous return to the systemic circulation (q LS ), and hepatic arterial inflow to the liver (q SL 24. The method of clause 24A or B, comprising the step of estimating

[0456] Article 26. Total hepatic inflow to the liver (Q L ) to the liver according to Equation 6A. SL ) and portal vein (q PL ) including both inflows: Q L =q SL +q PL ,(L minutes -1 ) Equation 6A During the ceremony, q SL is the hepatic arterial inflow velocity to the liver, q SL =0.25 Q L (L minutes -1 ) and q PL is the portal inflow rate to the liver, q PL =q SP (L minutes -1 ) and; q SP is the splanchnic artery blood flow velocity to the abdominal intestinal organs, q SP =0.75 Q L,init (L minutes -1 ) and optionally, Q LAn initial estimate for is approximately 1 L min -1 kg -1 Liver wet mass, Article 25 Method.

[0457] Article 27. Total hepatic venous return velocity to the systemic circulation (q LS ) is calculated by LS =q PL +q SL the methods set forth in clauses 25 or 26, including:

[0458] Article 28. Liver clearance (Cl H ) is estimated by Equation 7A: Cl H =Q L ·ER Equation 7A During the ceremony, ER is the extraction rate in the subject and is assumed to be 0.7; Any one of the methods set forth in Articles 24 to 27.

[0459] Clause 29. Obtaining subject-derived input data including blood or serum sample concentration data of an orally administered identifiable cholate compound collected from the subject at two time points within 180 minutes after oral administration, body mass index (BMI), hematocrit (Hct), and estimated volume of distribution (Vd) in the subject; fitting the input data to a trained function fitting neural network comprising a learning algorithm to generate a plurality of output points for distinguishable oral and intravenous cholate clearance curves; generating distinguishable oral and IV cholate clearance curves from the fitted data; AUC for objects involving trapezoidal numerical integrals Oral Value and AUC IV measuring the value; and AUCoral value and / or AUC IV calculating one or more indices of liver disease in the subject using the values, wherein the one or more indices are related to liver function in the subject. 1. A method for assessing liver function in a subject having or suspected of having or suffering from liver disease, comprising:

[0460] Clause 30. Obtaining subject-derived input data including blood or serum sample concentration data of an orally administered first identifiable cholate compound taken from the subject at two time points within 180 minutes after oral administration, blood or serum sample concentration data of an intravenously administered second identifiable cholate compound taken from the subject at one time point within 180 minutes after intravenous administration, an estimated volume of distribution (Vd) in the subject, and an estimated initial intravenous identifiable cholate concentration at 0 minutes of the intravenously administered second identifiable cholate compound based on Vd; fitting the input data to a trained function fitting neural network comprising a learning algorithm to generate a plurality of output points for distinguishable oral and intravenous cholate clearance curves; generating distinguishable oral and IV cholate clearance curves from the fitted data; AUC for objects involving trapezoidal numerical integrals Oral Value and AUC IV measuring the value; and AUCoral value and / or AUC IV calculating one or more indices of liver disease in the subject using the values, wherein the one or more indices are related to liver function in the subject. 1. A method for assessing liver function in a subject having or suspected of having or suffering from liver disease, comprising:

[0461] Article 31. The estimated Vd (L per kg body weight) in the subject is estimated by Equation 16A:

number

[0462] Clause 32. The method of any one of clauses 29 to 31, wherein the neural network is configured for a regression task and includes a two-layer feedforward network including a hidden layer and an output layer, optionally including a sigmoidal transformation function in the hidden layer and a linear transformation function in the output layer.

[0463] Clause 33. The method of any one of clauses 29 to 32, wherein the neural network including the learning algorithm is trained on a training dataset including a plurality of distinguishable oral and intravenous cholate clearance curves estimated by a non-compatmental minimal model (MM) from a combination of normal control subjects and patients with chronic liver disease.

[0464] Clause 34. The method of any one of clauses 29 to 33, wherein the learning algorithm is selected from the group consisting of Levenberg-Marquardt backpropagation, Bayesian regularization, BFGS quasi-Newton methods, resilient backpropagation, scaled conjugate gradient, conjugate gradient with Powell / Beale restart, Fletcher-Power conjugate gradient, Polak-Ribiere conjugate gradient, one-step secant method, variable learning rate gradient descent, gradient descent with momentum, and gradient descent learning algorithm.

[0465] Clause 35. The method of any one of clauses 29-34, wherein the output points include 5 minute increments from 0 to 180 minutes after oral administration, thereby resulting in 37 time points for each of the oral and IV clearance curves.

[0466] Clause 36.0: the initial intravenous identifiable cholate compound concentration at 0 minutes is Equation 16A:

number

[0467] Clause 37. (a) Dose of a first identifiable cholate compound to a subject oral ) and administering to the subject a dose of a second distinguishable cholate compound. iv receiving a plurality of blood or serum samples taken from the subject following simultaneous intravenous co-administration of the compounds of formula (I) and formula (II), wherein the samples are taken over a period of about 180 minutes or less, about 120 minutes or less, or about 90 minutes after administration; (b) quantifying the concentrations of the first and second distinguishable cholate compounds; and (c) generating individual subject oral and intravenous clearance curves from the concentrations of the first and second distinguishable cholate compounds, including using computer algorithm curve fitting to model the oral and intravenous clearance curves; and calculating the individualized areas under the oral and intravenous clearance curves (AUCoral) and (AUCiv) for the subject, respectively, wherein the multiple samples include blood or serum samples taken from the subject at at least five time points; and The construction of individual intravenous clearance curves is Equation 16A:

number

[0468] Article 38. One or more indices of liver disease are measured in the subject, including portal vein hepatic filtration fraction (HFRp), total body hepatic filtration fraction (HFRs), cholate SHUNT, liver disease severity index (DSI), and indexed hepatic reserve capacity (HR indexed ), and algebraic hepatic reserve (HR algebraic 38. The method of any one of clauses 1 to 37, wherein the compound is selected from the group consisting of:

[0469] Clause 39. The step of calculating one or more indices of liver disease comprises calculating the portal hepatic filtration rate (HFRp) in the subject according to Equation 10A:

number

[0470] Clause 40. The step of calculating one or more indices of liver disease comprises calculating systemic hepatic filtration rates (HFRs) according to Equation 12:

number

[0471] Clause 41. The method of any one of clauses 8 to 40, wherein calculating one or more indices of liver disease comprises estimating cholate SHUNT(F) according to Equation 13:

number

[0472] Clause 42. The step of calculating one or more indices of liver disease comprises estimating a Liver Disease Severity Index (DSI) according to Equation 14:

number

[0473] Clause 43. The step of calculating one or more indices of liver disease comprises: indexing hepatic reserve (HR) according to equation 15; indexed ) estimating:

number

[0474] Clause 44. The step of calculating one or more indices of liver disease comprises calculating algebraic hepatic reserve capacity (HR) according to Equation 15A. aldgebraic 43. The method of any one of clauses 39 to 42, comprising the step of estimating: HR algebraic =100-2·DSI Equation 15A.

[0475] Clause 45. The method of any one of clauses 1 to 44, wherein the first distinguishable cholate compound is a first stable isotope-labeled cholate compound, the optional second distinguishable cholate compound is a second stable isotope-labeled cholate compound, and the optional third distinguishable cholate compound is a third stable isotope-labeled cholate compound.

[0476] Clause 46. The first, optional second, and optional third stable isotope-labeled cholate compounds are d4-cholate, d2-cholate, d5-cholate, and 13 C-cholate, optionally d4-cholate is 2,2,4,4-d4 cholate, d5-cholate is 2,2,3,4,4-cholate, and further optionally 13C-cholate is 24- 13 46. ​​The method of clause 45, wherein C is cholate.

[0477] Clause 47. The method of any one of clauses 1 to 46, wherein the subject is a human subject.

[0478] Clause 48. Comparing one or more indices of liver disease in a subject with one or more cut-off values ​​as indicators of relative liver function in the subject. The method of any one of clauses 1 to 47, further comprising:

[0479] Clause 49. The method of clause 48, wherein the one or more cut-off values ​​are derived from one or more normal healthy controls, a group of known patients, or within subjects over time.

[0480] Article 50. Providing one or more indices of liver disease to a health care professional for the purpose of developing a treatment plan in a subject. 49. The method of any one of clauses 1 to 49, further comprising:

[0481] Clause 51. The method of clause 50, wherein one or more indices of liver disease are utilized for a purpose selected from the group consisting of determining the need for treatment, predicting response to treatment, predicting large esophageal varices, monitoring the effectiveness of treatment, individualizing dosing of one or more drugs, and predicting the risk of a clinical outcome in a subject.

[0482] Clause 52. The method of any one of clauses 1 to 51, wherein the liver disease is a chronic liver disease selected from the group consisting of chronic hepatitis C (CHC), chronic hepatitis B, metabolic dysfunction-related alcoholic liver disease (Met-ALD), alcoholic liver disease (ALD), fatty liver disease (SLD), fatty liver disease, alcoholic steatohepatitis (ASH), alcoholic hepatitis (AH), metabolic dysfunction-related fatty liver disease (MASLD), non-alcoholic fatty liver disease (NAFLD), steatosis, metabolic dysfunction-related steatohepatitis (MASH), non-alcoholic steatohepatitis (NASH), autoimmune liver disease, cryptogenic cirrhosis, hemochromatosis, Wilson's disease, alpha-1-antitrypsin deficiency, liver cancer, liver failure, cirrhosis, primary sclerosing cholangitis (PSC), and other cholestatic liver diseases.

[0483] Clause 53. The method of clause 51 or 52, wherein the clinical outcome is selected from the group consisting of Child-Turcotte-Pugh (CTP) progression, Model for End-Stage Liver Disease (MELD) progression, variceal bleeding, ascites, splenomegaly, varices, large esophageal varices, portal venous hypertension (PHTN), hepatic encephalopathy, hepatocellular carcinoma (HCC), decompensation, or liver-related death.

[0484] Clause 54. The method of any one of clauses 50 to 53, wherein the treatment is selected from the group consisting of antiviral therapy, antifibrotic therapy, antibiotics, immunosuppressive therapy, anticancer therapy, ursodeoxycholic acid, farnesoid X receptor ligands, insulin sensitizers, interventional therapy, liver transplantation, lifestyle modification, dietary restriction, low glycemic index diet, antioxidants, vitamin supplements, transjugular intrahepatic portosystemic shunting (TIPS), catheter-directed thrombolysis, balloon dilation and stent placement, balloon dilation and drainage, weight loss, exercise, and avoidance of alcohol.

[0485] Article 55. Monitoring the need for treatment in subjects Determining one or more indices of liver disease in the subject; and comparing one or more indices of liver disease to one or more cutoff values, wherein a change in one or more indices of liver disease compared to the cutoff values ​​indicates a need for treatment in the subject. Any one of clauses 1 to 54, including

[0486] Clause 56. The method of any one of clauses 49 to 55, wherein the known group of patients suffers from a disease or condition selected from the group consisting of chronic liver disease with a fibrotic stage; portal hypertension; Childs-Turcotte-Pugh (CTP) score A; CTP score B; CTP score C; Model for End-Stage Liver Disease (MELD) progression score, primary sclerosing cholangitis (PSC) not listed for transplant; PSC listed for liver transplant; PSC listed for liver transplant without varices; PSC listed for liver transplant with varices; ascites; stoma bleeding; splenomegaly; varices; large varices, variceal bleeding; hepatic encephalopathy, decompensation; or liver disease-related death.

[0487] Clause 57. The method of clause 56, wherein the fibrosis stage is determined by a method including liver biopsy or elastography.

[0488] Clause 58. The method of clause 57, wherein the liver biopsy determines an Ishak fibrosis score (liver biopsy) of F2 (mild portal fibrosis), F3, F4 (moderate bridging fibrosis), F5 (nodular and incomplete cirrhosis), or F6 (cirrhosis).

[0489] Article 59. Portal venous inflow to the liver (q PL 59. The method of any one of clauses 25 to 58, wherein said method differentiates between healthy controls, subjects without varicose veins, subjects with small varicose veins, and subjects with large varicose veins.

[0490] Table 26 Table 27 Table 28 Table 29 Table 30

Claims

1. obtaining blood or serum sample concentration data of the orally administered identifiable cholate compound collected from the subject at two time points after oral administration; measuring the area under the curve (AUCoral) of the blood or serum concentration of the orally administered distinguishable cholate compound in the subject; simulating a complete oral clearance curve using a compartmental model of oral cholate clearance, said compartmental model including the oral distinguishable cholate compound concentration data at the two time points, body mass index (BMI), body weight (BW), and optionally hematocrit (Hct) input values ​​for the subject; and calculating the area including the trapezoidal numerical integral to obtain the AUCoral; a measuring step comprising: calculating one or more indices of liver disease in the subject using the AUCoral, wherein the one or more indices are related to liver function in the subject.

1. A method for assessing liver function in a subject having or suspected of having or suffering from liver disease, comprising:

2. said step of obtaining concentration data of said orally administered distinguishable cholate compounds at said two time points comprising: receiving first and second blood or serum samples taken from the subject at first and second time points after a single oral dose of a first distinguishable cholate compound; and analyzing the sample to obtain the oral concentration data at the first and second time points. and optionally The blood or serum sample is collected within about 180 minutes, 120 minutes, 90 minutes, about 75 minutes, or about 60 minutes after the oral administration. The method of claim 1.

3. 3. The method of claim 2, wherein the first and second blood or serum samples are collected from the subject at least about 5 minutes to about 90 minutes, 10 minutes to 75 minutes, 20 minutes to 60 minutes, 25 minutes to 55 minutes, 30 minutes to 50 minutes, 35 minutes to 45 minutes, or about 40 minutes apart.

4. 4. The method of claim 3, wherein the first and second blood or serum samples are taken from the subject about 20 minutes and about 60 minutes, respectively, after the oral administration.

5. said step of obtaining concentration data of said orally administered distinguishable cholate compounds at said two time points comprising: receiving a single blood or serum sample taken from the subject after administering to the subject a first oral dose of a first distinguishable cholate compound and a second oral dose of a second distinguishable cholate compound; and analyzing said single sample to obtain oral concentration data for said first distinguishable cholate compound and said second distinguishable cholate compound at said two time points. and optionally the single blood or serum sample is obtained from the subject within about 180 minutes, 120 minutes, 90 minutes, about 75 minutes, or about 60 minutes after administration of the first oral dose; The method of claim 1.

6. 6. The method of claim 5, wherein the first and second oral doses are administered to the subject at least about 5 minutes to about 90 minutes, 10 minutes to 75 minutes, 20 minutes to 60 minutes, 25 minutes to 55 minutes, 30 minutes to 50 minutes, 35 minutes to 45 minutes, or about 40 minutes apart.

7. 6. The method of claim 5, wherein the single sample is taken from the subject about 20 minutes after the second oral dose and simultaneously about 60 minutes after the first oral dose.

8. estimating the area under the curve (AUCiv) of the blood or serum concentration of the intravenously administered distinguishable cholate compound; and calculating said one or more indices of liver disease in said subject using said AUCoral and AUCiv values; 8. The method of claim 1, further comprising:

9. The step of estimating the AUC comprises a linear regression model, and optionally, the linear regression model comprises Equation 11A: AUC IV =b 0 +b BW ・BW+β PO,20 ・C PO,20 +b PO,60 ・C PO,60 +b HFR,P ・HFR P Equivalent to 11A During the ceremony, β 0 is an intercept coefficient, optionally said intercept coefficient is 161.972; B BW is a weight coefficient, optionally wherein said weight coefficient is 0.6459; BW is the subject's body weight in kg; β PO,20 is the orally administered discriminative cholate concentration coefficient at the first time point, and optionally the β PO,20 is 16.9249; C PO,20 is the orally administered distinguishable cholate concentration at the first time point; β PO,60 is the orally administered discernible cholate concentration coefficient at the second time point, and optionally the β PO,60 is 89.2405; C PO,60 is the orally administered discernible cholate concentration at said second time point; β HFR,P is a portal vein HFR coefficient, optionally wherein said portal vein HFR coefficient is −0.4755; HFR P is the subject's measured portal vein HFR; The method of claim 8.

10. The step of estimating the AUCiv comprises: obtaining blood or serum sample concentration data of an intravenously administered third identifiable cholate compound in one of the samples taken from the subject; and exponentially fitting the intravenous concentration data to a systemic cholate clearance curve including rapid, moderate, and slow phases of clearance over a period of at least about 180 minutes following iv administration of the intravenous dose.

9. The method of claim 8, comprising:

11. 11. The method of claim 10, wherein the third distinguishable cholate is administered intravenously simultaneously with the first oral dose or at least about 5 to about 75 minutes, 10 to 70 minutes, 20 to 60 minutes, 25 to 55 minutes, 30 to 50 minutes, 35 to 45 minutes, or about 40 minutes after the first oral dose.

12. 11. The method of claim 10, wherein the blood or serum sample collected at a single time point is collected within about 90 minutes or less, 75 minutes or less, 60 minutes or less, 45 minutes or less, 30 minutes or less, or within about 20 minutes after the intravenous administration.

13. Rapid phase of the systemic cholate clearance curve (Y 0 ) is calculated according to Equation 20: [Equation 1] Equation 20 During the ceremony, t = time (0-20 min); C 0 is the initial concentration of the identifiable cholate compound administered intravenously; C 20 is the measured 20-minute concentration of the identifiable cholate compound administered intravenously; k fast is the elimination rate in the rapid phase estimated by Equation 18: [Equation 2] Equation 18 Optionally, C 0 is estimated according to equation 17: [Equation 3] Equation 17 During the ceremony, D IV is the intravenous dose of a third distinguishable cholate; BW is subject weight (kg); Vd is the volume of distribution (V) in L per kg body weight calculated according to Equation 16A d ) and: [Equation 4] Equation 16A where TPV is total plasma volume, BMI is body mass index, and Hct is hematocrit in the subject. The method of claim 10.

14. Systemic cholate clearance curve, rapid phase (Y 1 ) is calculated according to equation 21: [Equation 5] Equation 21 During the ceremony, t = time (20-45 min); k mod is the elimination rate during the optimal phase estimated by Equation 19: k mod =b 0,mod +b kfast ・k fast +b IV,20 ・C IV,20 +b PO,20,mod ・C PO,20 +b PO,60,mod ・C PO,60 Equivalent19 During the ceremony, β 0,mod is an intercept coefficient, optionally said intercept coefficient is 0.0268; β kfast is k fast coefficient, and optionally the k fast The coefficient is 0.546; β IV,20 is a coefficient of the intravenous discernible cholate concentration at the 20 minute time point; optionally, β IV,20 is 0.0045; β PO,20,mod is a coefficient of the oral distinguishable cholate concentration at the 20 minute time point, and optionally β PO,20,mod is -0.0007; β PO,60,mod is a coefficient of the oral distinguishable cholate concentration at the 60 minute time point, and optionally β PO,60,mod is -0.0052, 14. The method according to any one of claims 10 to 13.

15. The slow phase of the systemic cholate clearance curve (Y 2 ) is calculated according to equation 22: [Equation 6] Equation 22 During the ceremony, t = time (45-180 min); C 45 is the estimated 45-minute concentration of intravenously administered discernible cholate; k slow is the elimination rate in the slow phase estimated by the average value from multiple CLD patients, and optionally k slow is 0.018 minutes -1 That is, 15. The method according to any one of claims 10 to 14.

16. The area under each of the three exponential curve fits is calculated by trapezoidal numerical integration, and the AUC IV 16. The method of claim 10, wherein the values ​​of the input signal and output signal are summed to estimate

17. The compartmental model of oral clearance comprises: Compartment volumes of a plurality of compartments in the subject; and flow parameters between the compartments in the subject; 17. The method of claim 1, comprising estimating:

18. 18. The method of claim 17, wherein the compartmental model further comprises estimating cholate binding and dose administration in the subject.

19. 19. The method of claim 17 or 18, wherein the multiple compartments in the subject include the systemic, portal, and hepatic compartments.

20. estimating compartment volumes of the plurality of compartments; The whole body compartment volume (V S ), the portal vein compartment volume (V P ), and the liver compartment volume (V L ) 20. The method of claim 18 or 19, comprising: optionally each compartment volume is in liters (L).

21. The whole body compartment volume (V S ) is estimated according to equation 4A: V S =TBV・(1-Hct) Equation 4A During the ceremony, TBV is the total blood volume in the subject according to Equation 4: [Equation 7] Equation 4 During the ceremony, BMI is the body mass index (kg / m 2 ) and BW is the body weight (kg) of the subject; Hct is the hematocrit in the subject.

21. The method of claim 20.

22. The portal venous compartment volume (V P 22. The method of claim 20 or 21, wherein: V P =0.25・V S Equation 4B.

23. The liver compartment volume (V L ) is estimated according to equation 5A: V L =(0.275・22.46・BW・d L •f plasma Equation 5A (5 / 1000) In the formula, d L is 1.06 g mL -1 is the hypothetical liver tissue density of 23. The method of any one of claims 20 to 22.

24. the step of estimating the flow parameters between the plurality of compartments includes a system of first-order ordinary differential equations 1A, 2A, and 3; [Equation 8] Equation 1A; [Equation 9] Equation 2A; and [Equation 10] equation 3 In the formula, V is the total body (V S ), portal vein (V P ), and liver (V L ) is the volume of each of the compartments, and C is the total body (C S ), portal vein (C P ), and liver (C L ) is the concentration of orally administered distinguishable cholate compounds in each of the compartments, q is the flow rate between the compartments, and Cl H is the hepatic clearance and D PO,rate is the rate at which an orally administered distinguishable cholate compound enters the portal compartment; 24. The method of any one of claims 17 to 23.

25. the step of estimating the flow parameters between the plurality of compartments includes a system of first order ordinary differential equations 1B, 2B, and 3: [0011] Equation 1B; [0012] Equation 2B; and [0013] Equation 3, In the formula, V is the total body (V S ), portal vein (V P ), and liver (V L ) is the volume of each of the compartments, and C is the total body (C S ), portal vein (C P ), and liver (C L ) is the concentration of orally administered distinguishable cholate compounds in each of the compartments, q is the flow rate between the compartments, and Cl H is the hepatic clearance and D PO,rate is the rate at which an orally administered distinguishable cholate compound enters the portal compartment; 24. The method of any one of claims 17 to 23.

26. The step of estimating comprises determining the total hepatic inflow (Q L ), splanchnic arterial circulation (q SP ), hepatic portal vein inflow to the liver (q PL ), total hepatic venous return to the systemic circulation (q LS ), and hepatic arterial inflow to the liver (q SL 26. The method of claim 24 or 25, comprising estimating

27. The total hepatic inflow to the liver (Q L ) to the liver according to Equation 6A. SL ) and portal vein (q PL ) including both inflows: Q L =q SL +q PL ,(L·min -1 ) Equation 6A During the ceremony, q SL is the hepatic artery inflow velocity to the liver, q SL =0.25・Q L (L・minute -1 ) and; q PL is the portal vein inflow rate to the liver, q PL =q SP (L・minute -1 ) and; q SP is the splanchnic artery blood flow velocity to the abdominal intestinal organs, q SP =0.75・Q L,init (L・minute -1 ) and optionally, Q L Initial estimates for are approximately 1 L min -1 kg -1 Liver wet mass, 27. The method of claim 26.

28. The total hepatic venous return velocity to the systemic circulation (q LS ) is calculated by LS =q PL +q SL 28. The method of claim 26 or 27, comprising:

29. The liver clearance (Cl H ) is estimated by Equation 7A: Cl H =Q L ・ER Equation 7A During the ceremony, ER is the extraction ratio in the subject, assumed to be 0.7 for the identifiable cholate compounds; 29. The method of any one of claims 24 to 28.

30. obtaining input data derived from the subject, including blood or serum sample concentration data of the orally administered identifiable cholate compound taken from the subject at two time points within 180 minutes of oral administration, a body mass index (BMI), an estimated volume of distribution (Vd), and optionally a hematocrit (Hct) in the subject; fitting the input data to a trained function-fitting neural network comprising a learning algorithm to generate a plurality of output points for distinguishable oral and intravenous cholate clearance curves; generating distinguishable oral and IV cholate clearance curves from said fitted data; AUC for the above object including trapezoidal numerical integration Oral and AUC IV measuring the value; and AUCoral and / or AUC IV calculating one or more indices of liver disease in said subject using said values, wherein said one or more indices are related to liver function in said subject.

1. A method for assessing liver function in a subject having or suspected of having or suffering from liver disease, comprising:

31. obtaining input data derived from the subject, the input data including: (i) blood or serum sample concentration data of an orally administered first identifiable cholate compound taken from the subject at two time points within 180 minutes after oral administration; (ii) blood or serum sample concentration data of an intravenously administered second identifiable cholate compound taken from the subject at one time point within 180 minutes after intravenous administration; (iii) an estimated volume of distribution (Vd) in the subject; and (iv) an estimated initial intravenous identifiable cholate concentration at 0 minutes of the intravenously administered second identifiable cholate compound based on the Vd; fitting the input data to a trained function-fitting neural network comprising a learning algorithm to generate a plurality of output points for distinguishable oral and intravenous cholate clearance curves; generating distinguishable oral and IV cholate clearance curves from said fitted data; AUC for the above object including trapezoidal numerical integration Oral and AUC IV measuring the value; and AUCoral and / or AUC IV calculating one or more indices of liver disease in said subject using said values, wherein said one or more indices are related to liver function in said subject.

1. A method for assessing liver function in a subject having or suspected of having or suffering from liver disease, comprising:

32. The estimated Vd (L per kg body weight) for the subject is estimated by Equation 16A: [0014] Equation 16A where TPV is total plasma volume, BMI is body mass index, and Hct is hematocrit in the subject.

32. The method of claim 30 or 31.

33. 33. The method of any one of claims 30 to 32, wherein the neural network comprises a two-layer feedforward network configured for a regression task and including a hidden layer and an output layer, optionally including a sigmoidal transformation function in the hidden layer and a linear transformation function in the output layer.

34. 34. The method of any one of claims 30 to 33, wherein the neural network comprising the learning algorithm has been trained on a training dataset comprising a plurality of distinguishable oral and intravenous cholate clearance curves estimated by a non-compartmental minimal model (MM) from a combination of normal control subjects and patients with chronic liver disease.

35. 35. The method of any one of claims 30 to 34, wherein the learning algorithm is selected from the group consisting of Levenberg-Marquardt backpropagation, Bayesian regularization, BFGS quasi-Newton methods, resilient backpropagation, scaled conjugate gradient, conjugate gradient with Powell / Beale restart, Fletcher-Power conjugate gradient, Polak-Ribiere conjugate gradient, one-step secant method, variable learning rate gradient descent, gradient descent with momentum, and gradient descent learning algorithms.

36. 36. The method of any one of claims 30-35, wherein the output points comprise 1 to 10 minute, 2 to 8 minute, or 5 minute increments from 0 to 180 minutes after the oral administration, thereby resulting in 180 to 18, 60 to 23, or 37 time points for each of the oral and IV clearance curves.

37. said initial intravenous discernible cholate compound concentration at 0 minutes being: Equation 16A: [Equation 15] Equation 16A estimating Vd (L per kg body weight) in the subject by: V d Dividing the IV dose by the body weight (BW) It is estimated that 37. The method of any one of claims 31 to 36.

38. 1. A method for assessing liver function in a subject having or suspected of having or suffering from liver disease, comprising: (a) administering to said subject a dose of a first distinguishable cholate compound; oral and administering to said subject a dose of a second distinguishable cholate compound. iv receiving a plurality of blood or serum samples taken from the subject following simultaneous intravenous co-administration of (b) quantifying the concentrations of the first and second distinguishable cholate compounds; and (c) generating oral and intravenous clearance curves for an individual subject from the concentrations of said first and second distinguishable cholate compounds, comprising using a computer algorithm curve fitting to model the oral and intravenous clearance curves; and calculating the areas under the individualized oral and intravenous clearance curves (AUCoral) and (AUCiv) for said subject, respectively, wherein said plurality of samples comprises blood or serum samples taken from said subject at five or more time points. Including, Said generation of an individual intravenous clearance curve comprises: Equation 16A: [0016] Equation 16A estimating Vd (L per kg body weight) in the subject by: V d × body weight (BW) to obtain an initial estimated initial intravenous discernible cholate concentration. estimating an initial intravenous distinguishable cholate compound concentration in said subject at 0 minutes; and constructing said intravenous distinguishable cholate clearance curve including said estimated initial intravenous concentrations. Including, method.

39. The one or more indices of liver disease are portal vein hepatic filtration fraction (HFRp), systemic hepatic filtration fraction (HFRs), cholate SHUNT, Disease Severity Index (DSI), indexed hepatic reserve capacity (HR indexed ), and algebraic hepatic reserve (HR algebraic 39. The method of any one of claims 1 to 38, wherein the compound is selected from the group consisting of:

40. wherein said calculating one or more indices of liver disease comprises calculating a portal hepatic filtration rate (HFRp) in said subject according to Equation 10A: [Equation 17] Equation 10A In the formula, D PO is the oral dose of the orally administered distinguishable cholate, and BW is the body weight of the subject.

40. The method of claim 39.

41. wherein the step of calculating one or more indices of liver disease comprises calculating systemic hepatic filtration rates (HFRs) according to Equation 12: [Equation 18] Equation 12 In the formula, D IV is the administered intravenous oral dose of distinguishable cholate, and BW is the body weight of the subject.

40. The method of claim 39.

42. 42. The method of any one of claims 8 to 41, wherein the step of calculating one or more indices of liver disease comprises estimating cholate SHUNT(F) according to equation 13: [Equation 19] Equation 13.

43. wherein said calculating one or more indices of liver disease comprises estimating a Liver Disease Severity Index (DSI) according to Equation 14: [Equation 20] Equation 14 During the ceremony, HFR P,max is the upper limit of portal vein clearance from multiple healthy controls; HFR S,max is the upper limit of clearance from multiple healthy controls; A is a factor for scaling DSI from 0 to 50, 34. The method of any one of claims 31 to 33.

44. The step of calculating one or more indices of liver disease comprises indexing hepatic reserve (HR ) according to Equation 15. indexed ) estimating: [0000] Equation 15, During the ceremony, HFR P and HFR S was indexed to lean controls - 1 standard deviation (HFR P,lean and HFR S,lean ); A is a constant used to scale HR values ​​from 100 to 0.

44. The method of any one of claims 40 to 43.

45. The step of calculating one or more indices of liver disease may include calculating algebraic hepatic reserve (HR ) according to Equation 15A. aldgebraic 44. The method of any one of claims 40 to 43, comprising estimating: HR algebraic =100-2・DSI Equation 15A.

46. 11. The method of claim 10, wherein the first distinguishable cholate compound is a first stable isotope-labeled cholate compound, the optional second distinguishable cholate compound is a second stable isotope-labeled cholate compound, and the optional third distinguishable cholate compound is a third stable isotope-labeled cholate compound.

47. the first, optional second, and optional third stable isotope-labeled cholate compounds are d4-cholate, d5-cholate, d2-cholate, and 13 and optionally, the d4-cholate is 2,2,4,4-d4 cholate, the d5-cholate is 2,2,3,4,4-cholate, and further optionally, the 13C cholate is 24- 13 47. The method of claim 46, wherein the C-cholate is C-cholate.

48. The method of claim 1 , wherein the subject is a human subject.

49. comparing said one or more indices of liver disease in said subject with one or more cut-off values ​​as indicators of relative liver function in said subject.

49. The method of any one of claims 1 to 48, further comprising:

50. 50. The method of claim 49, wherein the one or more cutoff values ​​are derived from one or more normal healthy controls, a group of known patients, or within the subject over time.

51. providing said one or more indices of liver disease to a health care professional for purposes of formulating a treatment plan in said subject.

51. The method of any one of claims 1 to 50, further comprising:

52. 52. The method of claim 51, wherein the one or more indices of liver disease are utilized for a purpose selected from the group consisting of determining the need for treatment, predicting response to treatment, monitoring the effectiveness of treatment, predicting large esophageal varices, individualized drug dosing, and predicting the risk of a clinical outcome in the subject.

53. 53. The method of any one of claims 1 to 52, wherein the liver disease is a chronic liver disease selected from the group consisting of chronic hepatitis C (CHC), chronic hepatitis B, metabolic dysfunction-related alcoholic liver disease (Met-ALD), alcoholic liver disease (ALD), fatty liver disease (SLD), fatty liver disease, alcoholic steatohepatitis (ASH), alcoholic hepatitis (AH), metabolic dysfunction-related fatty liver disease (MASLD), non-alcoholic fatty liver disease (NAFLD), steatosis, metabolic dysfunction-related steatohepatitis (MASH), non-alcoholic steatohepatitis (NASH), autoimmune liver disease, cryptogenic cirrhosis, hemochromatosis, Wilson's disease, alpha-1-antitrypsin deficiency, liver cancer, liver failure, cirrhosis, primary sclerosing cholangitis (PSC), and other cholestatic liver diseases.

54. 53. The method of claim 52, wherein the clinical outcome is selected from the group consisting of Child-Turcotte-Pugh (CTP) progression, Model for End-Stage Liver Disease (MELD) progression, variceal bleeding, ascites, splenomegaly, varices, large esophageal varices, portal venous hypertension (PHTN), hepatic encephalopathy, hepatocellular carcinoma (HCC), decompensation, or liver-related death.

55. 53. The method of claim 52, wherein the treatment is selected from the group consisting of antiviral therapy, antifibrotic therapy, antibiotics, immunosuppressive therapy, anticancer therapy, ursodeoxycholic acid, farnesoid X receptor ligands, insulin sensitizers, interventional therapy, liver transplantation, lifestyle modifications, dietary restriction, a low glycemic index diet, antioxidants, vitamin supplements, transjugular intrahepatic portosystemic shunting (TIPS), catheter-directed thrombolysis, balloon dilation and stent placement, balloon dilation and drainage, weight loss, exercise, and alcohol avoidance.

56. monitoring the need for treatment in the subject; determining said one or more indices of liver disease in said subject; and comparing said one or more indices of liver disease with one or more cut-off values. Including, a change in said one or more indices of liver disease compared to a cutoff value indicates a need for treatment in said subject; 53. The method of claim 52.

57. The method of claim 50, wherein the known group of patients suffers from a disease or condition selected from the group consisting of chronic liver disease with a fibrotic stage; portal hypertension; Childs-Turcotte-Pugh (CTP) score A; CTP score B; CTP score C; Model for End-Stage Liver Disease (MELD) progression score, primary sclerosing cholangitis (PSC) not listed for transplant; PSC listed for liver transplant; PSC listed for liver transplant without varices; PSC listed for liver transplant with varices; ascites; stoma bleeding; splenomegaly; varices; large varices, variceal bleeding; hepatic encephalopathy, decompensation; or liver disease-related death.

58. 58. The method of claim 57, wherein the fibrosis stage is determined by a method comprising liver biopsy or elastography.

59. 59. The method of claim 58, wherein the liver biopsy determines an Ishak fibrosis score (liver biopsy) of F2 (mild portal fibrosis), F3, F4 (moderate bridging fibrosis), F5 (nodular and incomplete cirrhosis), or F6 (cirrhosis).

Images