Detection of liver protein to diagnose and treat liver injury

Abstract

A method of detecting and / or quantifying the presence of or increased levels of liver glycogen phosphorylase (Pygl) is disclosed. A sample is obtained from the blood of a subject, and intact Pygl, fragmented Pygl, and / or total is detected and / or quantified in the sample. Methods of diagnosing liver injury in a subject are also disclosed, wherein the presence of or increased levels of intact Pygl, fragmented Pygl, and / or total Pygl in blood is indicative of liver injury in the subject.

Description

[0001] DETECTION OF LIVER PROTEIN TO DIAGNOSE AND TREAT LIVER INJURY CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 759,799, filed on February 18, 2025, the content of which is incorporated herein by reference in its entirety.

[0003] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING A Sequence Listing accompanies this application and is submitted as an XML file of the sequence listing named “16985200196_SL. xml” which is 4,491 bytes in size and was created on February 18, 2026. The sequence listing is electronically submitted via Patent Center with the application and is incorporated herein by reference in its entirety.

[0004] INTRODUCTION

[0005] Suspected liver injury is one of the most common reasons for termination of new drugs during clinical trials. However, identifying drug-induced liver injury (DILI) during clinical trials is a challenge, and there is concern that many drugs terminated due to potential liver injury are actually safe, causing losses to pharmaceutical companies and depriving patients of effective treatments. Currently, the US FDA and pharmaceutical companies rely largely on plasma alanine aminotransferase (ALT) elevations as a signal of DILI. Elevated ALT is considered a serious concern and can lead to termination of drug development. Typically, ALT is released from dying cells and therefore reflects tissue injury. However, the FDA itself acknowledges that ALT elevations are not specific for liver damage. In fact, a number of generally safe drugs are known to cause even large ALT elevations in some people, with no other evidence of DILI. Therefore, a need exists for new biomarkers and methods with greater specificity for detecting liver injury.

[0006] SUMMARY

[0007] A method of detecting the presence of or increased levels of liver glycogen phosphorylase (Pygl) is provided herein. The method comprises obtaining a blood sample from a subject and detecting the presence of or increased levels of intact Pygl, fragmented Pygl, and / or total Pygl, relative to a control. The presence of Pygl in the a blood or serum sample from a subject or increased Pygl levels in a blood or serum sample from the subject may be associated with liver injury, including DILI. The Pygl may be detected using mass spectrometry orimmunoassay. The immunoassay used may be a Western blot, an enzyme-linked immunosorbent assay (ELISA), or a lateral flow assay (LFA).

[0008] Further, methods of diagnosing liver injury in a subject based on detection of or increased levels of intact Pygl, fragmented Pygl, and / or total Pygl are provided. The methods may further comprise treating the subject for liver injury, including via administration of N-acetylcysteine.

[0009] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a drawing illustrating a mechanism of protein fragmentation following liver injury. Dying cells release proteases into the necrotic milieu, which then cleave proteins in an unregulated manner.

[0010] FIG. 2 demonstrates the results from a mouse model of acetaminophen-induced liver injury. Mice were treated with acetaminophen (250 mg / kg; to induce liver injury), dexamethasone (100 mg / kg; to induce non-liver injury related elevation of serum alanine aminotransferase), or vehicle control. Mass spectrometry analysis revealed Pygl fragments of approximately 40-50 kDa only in mice treated with acetaminophen.

[0011] FIG. 3 is a photograph of an immunoblot from the sera of either patients with liver injury caused by acetaminophen (APAP) overdose or healthy controls (HC) demonstrating detection of intact Pygl and Pygl fragments in the samples from those with liver injury.

[0012] FIG. 4 is a drawing illustrating an ELISA method for quantification of fragmented Pygl in a sample collected from the blood of a subject.

[0013] FIG. 5 depicts ELISA results showing an increase in serum total Pygl levels in humans (left) and mice (right) with APAP-induced liver injury. HV=healthy volunteers; ALF=acute liver failure caused by APAP overdose.

[0014] DETAILED DESCRIPTION

[0015] Suspected liver injury is the 4thmost common reason for termination of new drugs during clinical trials. Currently, the FDA and pharmaceutical companies largely rely on measurement of plasma ALT levels to test for DILI. However, ALT lacks specificity for liver injury. False positives are known, potentially causing losses to pharmaceutical companies and depriving patients of new and promising treatments. Thus, development of methods with improved specificity for detecting liver injury is needed.Proteases are enzymes that cleave other proteins. Under normal conditions, proteases are tightly regulated to prevent the destruction of proteins that are necessary for cell structure and function. However, it has been demonstrated that, when cells in the liver are damaged, regulation of proteases is lost. (Limaye et al., Toxicol Appl Pharmacol., 2003). Evidence shows that dying cells release proteases into the necrotic milieu, where they are free to cleave proteins in an unregulated manner (FIG. 1). As a result, some proteins that are not normally targeted by proteases become fragmented.

[0016] In the examples, we induced liver injury using acetaminophen overdose and evaluated the effects on serum levels of liver proteins by proteomic mass spectrometry. Fragmented liver proteins, including liver Pygl, were detected only in the acetaminophen treatment group. Pygly was actually only observed in serum samples from subjects with liver injury. We further evaluated sera from patients with liver injury by western blot and detected Pygl and Pygl fragments. Subjects without liver injury did not have Pygl or Pygl fragments apparent in the sera. This was true even under conditions that induce ALT release but are not associated with liver injury as shown in the Examples. In addition, as compared to control animals or healthy volunteers, we detected increased serum levels of total Pygl in mice and humans following acetaminophen overdose. Collectively, these results demonstrate that liver injury is associated with elevated levels of particular liver proteins and fragmented liver proteins including Pygl and elucidate a method of detecting and / or diagnosing liver injury in a subject by detecting the presence of or increased levels of Pygl in the sera of subjects.

[0017] Thus, methods for detecting and / or quantifying the presence of Pygl in the serum and increased levels of liver Pygl in a sample obtained from a subject and methods for diagnosing liver injury in a subject based on the presence of increased levels of Pygl are provided herein. The subject may be a human being or an animal from any class including but not limited to Mammalia (mammals), any order including but not limited to Rodentia, and any genus including but not limited to Canis (dogs), Felis (cats), Bos (cows), Equus (horses), Sus (pigs).

[0018] In the methods, the sample is a biological sample collected from the blood of a subject. The sample may be obtained directly from the subject (e.g., by blood sample) or from a third party (e.g., received from an intermediary, such as a healthcare provider or lab technician). The sample may be whole blood, serum, or plasma. The sample may contain the fragmented liver protein.In embodiments, the methods comprise detecting the presence of or increased levels of Pygl in the sample. The detecting may comprise detecting the presence of or increased levels of intact Pygl, fragmented Pygl, and / or total Pygl. As used herein, “fragmented” liver protein or liver protein “fragments” refer to polypeptides produced by cleavage of an intact protein that is expressed in the liver of the subject, “intact” protein refers to a protein which has not undergone said cleavage, and “total” protein includes both fragmented and intact protein. The cleavage of the intact protein may be caused by a protease enzyme. The fragmented liver protein may include without limitation Pygl fragments. The fragmented liver protein is Pygl and the fragmented Pygl has a molecular weight of between about 40 kDa to about 70 kDa. In a further embodiment, the fragmented Pygl has a molecular weight of about 50 kDa. The increased levels comprise intact Pygl, fragmented Pygl, and / or total Pygl may be detected relative to a control. As used herein, a “control” for measuring the level of Pygl may refer to the level of intact Pygl, fragmented Pygl, and / or total Pygl in a sample from one or more non-diseased patient, a positive control, or a negative control with a known amount of intact Pygl, fragmented Pygl, and / or total Pygl, or may refer to a first sample taken from the same patient from an earlier timepoint that can be used to monitor the change in levels of intact Pygl, fragmented Pygl, and / or total Pygl over time. As shown in the Examples, Pygl is generally not detected in the serum of healthy individuals so detection of the presence of Pygl in the serum at all may be sufficient the diagnose an individual with liver injury and begin treatment for liver injury or stop any treatment with a drug suspected of inducing liver injury.

[0019] The sample may be processed prior to detection to remove unwanted molecules and / or proteins in order to improve signal detection. Those of skill in the art are aware of multiple processing techniques to remove such unwanted molecules and / or proteins, including centrifugation, precipitation, and affinity chromatography techniques. In one embodiment, the sample is processed in order to remove at least a portion of the albumin and immunoglobulin from the sample. The albumin and immunoglobulin may be removed via affinity chromatography techniques using Cibacron Blue beads or anti-albumin and / or anti-immunoglobulin antibodies.

[0020] The liver protein in the sample may be detected using any suitable detection method. When fragmented liver protein is detected, the detection method may be capable of distinguishing between fragmented and intact liver protein. Suitable detection methods for performing the disclosed methods, include, without limitation, mass spectrometry-based proteomics,immunoassay, or lateral flow assay. The immunoassay used may be a western blot, an ELISA, or a lateral flow assay. In an embodiment, the fragmented Pygl is detected by western blot using an antibody capable of binding at least to fragmented Pygl. The antibody may also be capable of binding to intact Pygl. The antibody may be a rabbit monoclonal antibody which binds to mouse or human Pygl (e.g., PYGL (E4O1P) Rabbit mAb #42103, Cell Signaling Technologies). The presence and detection of any amount of fragmented Pygl in a sample from a subject may be indicative of liver injury. Thus quantification may not be needed or required. If quantified, the amount of fragmented Pygl in the sample may represent 0.001%, 0.1%, 1%, 2%, 3%, 5%, 8%, 10%, 15%, 20% or even 25% of the total Pygl detected in a sample.

[0021] In some embodiments, the intact Pygl, fragmented Pygl, and / or total Pygl is detected and optionally quantified via an enzyme-linked immunosorbent assay (ELISA). In one embodiment, a capture antibody that is affixed to a solid-surface binds to an epitope of fragmented Pygl in the sample, immobilizing the fragmented Pygl. The capture antibody may be specific for the fragmented Pygl. An antibody specific for fragmented Pygl may recognize epitopes not available in full-length intact Pygl or may recognize a terminus near the cleavage site not available in the intact Pygl. Next, a detection antibody that is capable of generating a detectable signal is added that binds to an epitope on the immobilized, fragmented Pygl. In other embodiments, the capture antibody may bind to an epitope present on both fragmented and intact Pygl. In these embodiments, the detection antibody may be specific for the fragmented Pygl.

[0022] In a further embodiment, two ELIS As are employed (FIG.4) to detect the fragmented Pygl. In the first ELISA, total Pygl levels — including both intact and fragmented protein — are detected and / or quantified. In the second ELISA, only intact Pygl is detected and / or quantified. These double ELISA assays then allow for calculation of the presence of fragmented Pygl by the difference in the total Pygl level as compared to the intact Pygl level. As one option a capture antibody and first detection antibody that bind to epitopes near the same terminus of the Pygl. As used herein, terminus refers to either end (N-terminus or C-terminus) of the Pygl protein. Intact Pygl has both termini, while fragmented Pygl has only one terminus. In the second ELISA, intact Pygl is quantified using a capture antibody that binds to an epitope near one terminus and a second detection antibody that binds to an epitope near the opposite terminus. Thus, the capture antibody binds to both intact and fragmented Pygl, while the detection antibody only binds to the intact Pygl. Because both termini must be present to obtain a detectable signal, only intact protein isquantified. Finally, the levels of fragmented Pygl in the sample are quantified by subtracting intact Pygl levels from total Pygl levels (Total-Intact=Fragmented). For this ELISA the capture and detection antibodies for detecting the total Pygl may both recognize epitopes on the N- or C-terminal half of the Pygl protein, e.g. the same portion of Pygl. The two antibodies may recognize epitopes that are less then 200 amino acids apart, less than 150 amino acids apart, less than 100 amino acids apart or less than 50 amino acids apart. In contrast, the antibodies for detection of intact Pygl in the second ELISA are specific for epitopes on opposite halves of the intact Pygl protein, e.g. the antibodies do not bind the same portion of Pygl. In other words, the capture antibody may bind to an epitope on the N-terminal half of Pygl and the detection antibody may bind to an epitope on the C-terminal half of Pygl. The Pygl detected by the ELISA would represent only full-length or intact Pygl. In this ELISA, the epitopes of the capture and detection antibody are more than 100 amino acids apart, more than 150 amino acids apart, more than 200 amino acids apart. The epitopes recognized by the capture and detection antibodies may be on either side of the protease cleavage site of Pygl once the protease cleavage site is defined.

[0023] In another embodiment using two ELISAs to detect fragmented Pygl, the levels of total Pygl are determined in a first ELISA. The first ELISA comprises a first capture antibody that binds to both intact and fragmented Pygl and a first detection antibody that binds to both intact and fragmented Pygl. The second ELISA comprises at least one second capture antibody or at least one second detection antibody that binds to intact Pygl but does not bind to fragmented Pygl. In all of the two ELISA assay methods the levels of fragmented Pygl are determined by subtracting the intact Pygl levels from the total Pygl levels. Those of skill in the art will readily appreciate that the ELISAs described above may also performed individually to detect and / or quantify intact or total Pygl levels.

[0024] Those of skill in the art are aware of multiple techniques to generate and measure a detectable signal in an ELISA, including without limitation direct fluorescence detection via fluorophore-labeling of the detection antibody, indirect fluorescence detection via fluorophorelabeling of a secondary antibody which binds to the detection antibody, direct chromogenic detection via conjugation of the detection antibody to an enzyme that produces a chromogenic signal, indirect chromogenic detection via conjugation of the secondary antibody to an enzyme that produces a chromogenic signal, and indirect signal amplification using an avidin-biotin complex method or labeled streptavidin-biotin method. ELISAs can be quantitative or qualitativeand those of skill in the art are aware of means to quantitate an ELISA using fluorescent or chromogenic readout systems.

[0025] In some embodiments, the intact Pygl, fragmented Pygl, and / or total Pygl is detected by lateral flow assay (LFA) using a lateral flow device. As used herein, a “lateral flow device” is a porous device capable of detecting the presence of a target analyte traversing one or more beds. Lateral flow devices typically comprise (a) an aqueous sample loading area at one end; (b) an area comprising a capture agent comprising a first binding agent immobilized on a substrate; (c) an area comprising a reporter comprising a detectable label conjugated to a second binding agent, wherein the reporter is not bound to the lateral flow device and is capable of wi eking across the lateral flow device; and (d) absorbent material, wherein the absorbent material wicks an aqueous sample across the lateral flow device when the aqueous sample is added to the sample loading area. In embodiments that utilize a lateral flow device, the assay results may be displayed using LFA strips that may provide a readout of one or more identifiable marks, such as a test or control line. The strips comprise the capture agent in a region referred to as the “test area”. The test area can be any shape with well-defined boundaries, such as a dot or a line.

[0026] In some embodiments, the capture agent is an immobilized capture antibody that binds to an epitope of fragmented Pygl in the sample, immobilizing the fragmented Pygl. The capture antibody may be specific for the fragmented Pygl (e.g., may recognize epitopes not available in full-length intact Pygl or may recognize a terminus near the cleavage site not available in the intact Pygl). In these embodiments, the reporter may be a detection antibody that binds to an epitope on the immobilized, fragmented Pygl.

[0027] In other embodiments, the capture agent may be an immobilized capture antibody that binds to an epitope present on both fragmented and intact Pygl (e.g., near one terminus of the Pygl protein), thereby immobilizing both the fragmented and intact Pygl. In these embodiments, the reporter may be a detection antibody that is specific for the fragmented Pygl.

[0028] In another embodiment, two LFAs and / or LFA strips are employed to detect the fragmented Pygl. In the first LFA, total Pygl levels are detected and / or quantified. In the second LFA, only intact Pygl is detected and / or quantified, allowing for calculation of the presence of fragmented Pygl by the difference in the total Pygl level as compared to the intact Pygl level. In these embodiments, the capture agent may be an immobilized capture antibody that binds to an epitope near one terminus of the Pygl protein, thereby immobilizing both the fragmented and intactPygl. In the first LFA, the reporter may be a detection antibody that binds to an epitope near the same terminus as the capture antibody, thereby allowing for the detection and / or quantification of total Pygl levels. In the second LFA, the reporter may be a detection antibody that binds to an epitope near the opposite terminus as the capture antibody, thereby allowing for the detection and / or quantification of intact Pygl levels. Finally, the levels of fragmented Pygl in the sample may be quantified by subtracting intact Pygl levels from total Pygl levels (Total-Intact=F ragmented) .

[0029] In another embodiment using two LFAs and / or LFA strips to detect fragmented Pygl, levels of total Pygl may be determined in a first LFA comprising a first capture agent (e.g., a first capture antibody) that binds to both intact and fragmented Pygl and a first reporter (e.g., a first detection antibody) that binds to both intact and fragmented Pygl. The second LFA may then comprise at least one second capture agent (e.g., a second capture antibody) or at least one second reporter (e.g., a second detection antibody that binds to intact Pygl but does not bind to fragmented Pygl. In all of the two LFA assay methods the levels of fragmented Pygl are determined by subtracting the intact Pygl levels from the total Pygl levels. A skilled artisan will readily understand the considerations relevant for identifying suitable detection and / or capture agents / antibodies, including those described above with respect to ELISA, and that the LFAs described above may also be performed individually to detect and / or quantify intact or total Pygl levels.

[0030] Those of skill in the art are aware of multiple techniques to generate and measure a detectable signal using LFA, including, without limitation, fluorescence detection via fluorophorelabeling of the detection antibody, indirect fluorescence detection via fluorophore-labeling of a secondary antibody which binds to the detection antibody, direct colorimetric detection via colorimetric labeling (e.g., a gold nanoparticle label) of the detection antibody, indirect colorimetric detection via colorimetric labeling of a secondary antibody which binds to the detection antibody, and indirect signal amplification using an avidin-biotin complex method or labeled streptavidin-biotin method.

[0031] In some embodiments, the lateral flow device is configured such that detection is accomplished by visual inspection, either with or without additional instrumentation. For example, results can be quantified by imaging and analysis with a computer. The results can be scanned with a smartphone and electronically sent to a clinician, e.g., using an Adobe Acrobat grayscaleconverter or an ImageJ image processing software to quantify the visible light signal from a gold nanoparticle. Likewise, a color wheel for visualization of positive tests may be utilized.

[0032] In some embodiments, the presence of or increased levels of Pygl in the sample are associated with or caused by a liver injury in the subject. As used herein, liver injury refers to a condition in which the cells of the liver are damaged due to external or internal factors. The liver injury may be, without limitation, DILI, ischemic / hypoxic hepatitis, viral hepatitis, alcohol-induced liver injury, or liver injury due to steatosis (fatty liver) The DILI may be either expected or idiosyncratic. As used herein, expected DILI is an injury caused by direct liver toxicity of a drug and is generally dose-dependent and amenable to translational animal models. Idiosyncratic DILI is an injury caused by a drug that is unexpected based on the drug’s mechanism of action, is less dose-dependent, and is typically difficult to predict based on animal toxicology models. Idiosyncratic DILI may have a longer latency than expected DILI. Therefore, in order to detect liver proteins associated with expected or idiosyncratic DILI, samples may be collected from a subject at various intervals. For example, samples may be collected at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 3 days, 1 week, 2 weeks, 1 month, 2 months, or 3 months after administration of a drug. If a drug is administered using a sub-chronic or chronic dosing regimen (e.g., as part of a clinical trial), samples may be collected daily, weekly, biweekly, or monthly during or after the period of drug administration to monitor for the presence of fragmented liver proteins as a means for detection of DILI.

[0033] Methods of diagnosing liver injury in a subject are also disclosed. In an aspect, the method of diagnosing liver injury in a subject includes detecting and / or quantifying intact Pygl, total Pygl, and / or fragmented Pygl in the subject. The intact Pygl, total Pygl, and / or fragmented Pygl may be detected and / or quantified using any of the methods disclosed herein, including without limitation mass spectrometry, western blot, ELISA, and LFA. In a further aspect, the presence of and / or increased levels intact Pygl, total Pygl, and / or fragmented Pygl in the subject’s blood is indicative of liver injury in the subject. The liver injury may be indicated by detected fragmented Pygl levels that comprise at least 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or at least 90% of the total Pygl levels in the subject’s blood.

[0034] In some embodiments, the methods further comprise treating the subject for liver injury. Suitable treatments for liver injury are well-known in the art and include, without limitation,antibiotics / antivirals, activated charcoal, corticosteroids, immunosuppressants, and N-acetylcysteine. In some embodiments, the treatment comprises administering N-acetylcysteine.

[0035] The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and / or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements. The terms “a”, “an” and “the” may mean one or more than one unless specifically delineated.

[0036] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values verynear to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0037] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims. All references, including patents, patent publications and non-patent literature, cited herein are hereby incorporated by reference in their entirety. Any conflict between statements in references and those made herein should be resolved in favor of the statements contained herein.

[0038] EXEMPLARY EMBODIMENTS

[0039] Embodiment 1. A method of detecting fragmented glycogen phosphorylase (Pygl) in a subject, comprising

[0040] a. obtaining a sample from the blood of a subject;

[0041] b. detecting fragmented Pygl in the sample.

[0042] Embodiment 2. The method of embodiment 1, wherein fragmented Pygl in the blood is associated with liver injury in the subject.

[0043] Embodiment 3 .The method of embodiment 1 or 2, wherein the sample is a serum or plasma sample.

[0044] Embodiment 4. The method of any one of the preceding embodiments, wherein at least a portion of the albumin is removed from the sample prior to step (b).

[0045] Embodiment 5. The method of any one of the preceding embodiments, wherein the liver injury is drug-induced liver injury.

[0046] Embodiment 6. The method of embodiment 5, wherein the sample is collected at least six hours after administration of a drug.

[0047] Embodiment 7. The method of embodiment 6, wherein the sample is collected no more than 3 months after administration of a drug.

[0048] Embodiment 8. The method of any one of the preceding embodiments, wherein the method may be repeated at regular intervals during or after administration of a drug.

[0049] Embodiment 9. The method of embodiment 8, wherein the regular intervals are weekly, biweekly or monthly.Embodiment 10. The method of any one of the preceding embodiments, wherein the subject is a mammal.

[0050] Embodiment 11. The method of embodiment 10, wherein the mammal is selected from the group consisting of a rodent (mouse or rat), a cat, a dog, a cow, a horse, a pig and a human.

[0051] Embodiment 12. The method of any one of the preceding embodiments, wherein the fragmented Pygl is detected using mass spectrometry.

[0052] Embodiment 13. The method of any one of the preceding embodiments, wherein the fragmented Pygl is detected using an immunoassay.

[0053] Embodiment 14. The method of embodiment 13, wherein the immunoassay is a western blot using an antibody capable of binding to at least a fragmented Pygl.

[0054] Embodiment 15. The method of embodiment 13, wherein the immunoassay is an enzyme-linked immunosorbent assay (ELISA).

[0055] Embodiment 16. The method of any one of embodiments 1-11, wherein the fragmented Pygl is detected using a lateral flow assay (LFA)

[0056] Embodiment 17. The method of embodiment 15 or 16, wherein the method comprises an antibody that binds to an epitope of the fragmented Pygl and does not bind to intact Pygl.

[0057] Embodiment 18. The method of embodiment 15 or 16, wherein step (b) further comprises:

[0058] i) determining the levels of total Pygl in a first ELISA or LFA, the ELISA or LFA comprising a first capture antibody that binds to both intact and fragmented Pygl and a first detection antibody that binds to both intact and fragmented Pygl;

[0059] ii) determining the levels of intact Pygl in a second ELISA or LFA, the ELISA or LFA comprising at least one second capture antibody or at least one second detection antibody that binds to intact Pygl, but does not bind to fragmented Pygl; and

[0060] iii) quantifying the levels of fragmented Pygl by subtracting the intact Pygl levels from step ii) from the total Pygl levels of i).

[0061] Embodiment 19. The method of embodiment 15, wherein step (b) further comprises: i) determining the levels of total Pygl in a first ELISA or LFA, the ELISA or LFA comprising a first capture antibody and a first detection antibody which are both capable of binding to the same portion of Pygl;ii) determining the levels of intact Pygl in a second ELISA or LFA, the ELISA or LFA comprising a second capture antibody and a second detection antibody that do not bind to the same portion of Pygl; and

[0062] iii) quantifying the levels of fragmented Pygl by subtracting the intact Pygl levels from step ii) from the total Pygl levels of i).

[0063] Embodiment 20. A method of diagnosing liver injury in a subject comprising detecting fragmented Pygl using the method of any one of the preceding embodiments, wherein the presence of fragmented Pygl in the blood is indicative of liver injury in the subject.

[0064] Embodiment 21. The method of embodiment 20, wherein the liver injury is drug-induced liver injury.

[0065] Embodiment 22. The method of embodiment 20, wherein the liver injury is indicated by fragmented Pygl levels that comprise at least 5% of the total Pygl detected in the sample.

[0066] Embodiment 23. A method of detecting increased levels of glycogen phosphorylase (Pygl) in a subject, comprising

[0067] a. obtaining a sample from the blood of a subject;

[0068] b. detecting increased levels of Pygl in the sample, wherein the increased levels of Pygl (i) are increased relative to a control and (ii) comprise intact Pygl, fragmented Pygl, and / or total Pygl.

[0069] Embodiment 24. The method of embodiment 23, wherein the increased levels of Pygl in the blood is associated with liver injury in the subject.

[0070] Embodiment 25. The method of embodiment 23 or 24, wherein the sample is a serum or plasma sample.

[0071] Embodiment 26. The method of any one of embodiments 23-25, wherein at least a portion of the albumin is removed from the sample prior to step (b).

[0072] Embodiment 27. The method of any one of embodiments 23-26, wherein the liver injury is drug-induced liver injury.

[0073] Embodiment 28. The method of embodiment 27, wherein the sample is collected at least six hours after administration of a drug.

[0074] Embodiment 29. The method of embodiment 28, wherein the sample is collected no more than 3 months after administration of a drug.Embodiment 30. The method of any one of any one of embodiments 23-29, wherein the method may be repeated at regular intervals during or after administration of a drug.

[0075] Embodiment 31. The method of embodiment 30, wherein the regular intervals are weekly, biweekly or monthly.

[0076] Embodiment 32. The method of any one of any one of embodiments 23-31, wherein the subject is a mammal.

[0077] Embodiment 33. The method of embodiment 32, wherein the mammal is selected from the group consisting of a rodent (mouse or rat), a cat, a dog, a cow, a horse, a pig and a human.

[0078] Embodiment 34. The method of any one of embodiments 23-33, wherein the increased levels of Pygl is detected using mass spectrometry.

[0079] Embodiment 35. The method of any one of embodiments 23-34, wherein increased levels of Pygl is detected using an immunoassay

[0080] Embodiment 36. The method of embodiment 35, wherein the immunoassay is a western blot using an antibody capable of binding to at least a fragmented Pygl.

[0081] Embodiment 37. The method of embodiment 35, wherein the immunoassay is an enzyme-linked immunosorbent assay (ELISA).

[0082] Embodiment 38. The method of any one of embodiments 23-34, wherein the increased levels of Pygl is detected using a lateral flow assay (LFA).

[0083] Embodiment 39. The method of embodiment 37, wherein step (b) further comprises determining the levels of total Pygl, and wherein the ELISA comprises a capture antibody that binds to both intact and fragmented Pygl and a detection antibody that binds to both intact and fragmented Pygl.

[0084] Embodiment 40. The method of embodiment 37, wherein step (b) further comprises determining the levels of intact Pygl, and wherein the ELISA comprises at least one capture antibody or at least one detection antibody that binds to intact Pygl, but does not bind to fragmented Pygl.

[0085] Embodiment 41. The method of embodiment 37, wherein step (b) further comprises: i) determining the levels of total Pygl in a first ELISA, the ELISA comprising a first capture antibody that binds to both intact and fragmented Pygl and a first detection antibody that binds to both intact and fragmented Pygl;ii) determining the levels of intact Pygl in a second ELISA, the ELISA comprising at least one second capture antibody or at least one second detection antibody that binds to intact Pygl, but does not bind to fragmented Pygl; and

[0086] iii) quantifying the levels of fragmented Pygl by subtracting the intact Pygl levels from step ii) from the total Pygl levels of i).

[0087] Embodiment 42. The method of embodiment 37, wherein step (b) further comprises determining the levels of total Pygl, and wherein the ELISA comprises a capture antibody and a detection antibody which are both capable of binding to the same portion of Pygl.

[0088] Embodiment 43. The method of embodiment 37, wherein step (b) further comprises determining the levels of intact Pygl, and wherein the ELISA comprises a capture antibody and a detection antibody that do not bind to the same portion of Pygl.

[0089] Embodiment 44. The method of embodiment 37, wherein step (b) further comprises: i) determining the levels of total Pygl in a first ELISA, the ELISA comprising a first capture antibody and a first detection antibody which are both capable of binding to the same portion of Pygl;

[0090] ii) determining the levels of intact Pygl in a second ELISA, the ELISA comprising a second capture antibody and a second detection antibody that do not bind to the same portion of Pygl; and iii) quantifying the levels of fragmented Pygl by subtracting the intact Pygl levels from step ii) from the total Pygl levels of i).

[0091] Embodiment 45. A method of diagnosing liver injury in a subject comprising detecting increased levels of fragmented Pygl using the method of any one of the preceding embodiments, wherein the increased levels of fragmented Pygl in the blood is indicative of liver injury in the subject.

[0092] Embodiment 46. A method of diagnosing liver injury in a subject comprising detecting increased levels of intact Pygl using the method of any one of embodiments 23-44, wherein the increased levels of intact Pygl in the blood is indicative of liver injury in the subject, and wherein the increased levels of intact Pygl are increased relative to a control.

[0093] Embodiment 47. A method of diagnosing liver injury in a subject comprising detecting increased levels of total Pygl using the method of any one of embodiments 23-44, wherein the increased levels of total Pygl in the blood is indicative of liver injury in the subject, and wherein the increased levels of total Pygl are increased relative to a control.Embodiment 48. The method of any one of embodiments 23-44, wherein the liver injury is drug-induced liver injury.

[0094] Embodiment 49. The method of any one of embodiments 23-44, further comprising treating the subject for the liver injury.

[0095] Embodiment 50. The method of embodiment 49, wherein the treatment comprises administering N-acetylcysteine.

[0096] EXAMPLES

[0097] Example I: Identification of Liver Protein Fragments after Liver Injury

[0098] To determine if liver injury would result in detectable fragments of liver proteins in circulation, we employed a mouse model of acetaminophen (APAP) overdose. Mice were treated with either APAP (250 mg / kg) to induce liver injury, dexamethasone (Dex; 100 mg / kg), or vehicle control (saline or DMSO). The Dex treatment group was included to induce elevation of serum ALT without liver injury, mimicking what is sometimes seen in clinical trials, where generally safe drugs are known to elevate ALT concentrations with no other evidence of liver damage. Collectively, these treatment groups allowed us to identify protein fragments that are elevated in blood only when there is a true injury (e.g., the APAP treatment).

[0099] Serum was collected from the mice at 6 hours post-APAP or saline treatment and 24 hours post-Dex or DMSO treatment, and serum proteins were separated by gel electrophoresis. Bands were excised from the gel at regular intervals along each sample and subjected to untargeted mass spectrometry-based proteomics to identify peptides. Bands below albumin were excised in order to avoid albumin contamination.

[0100] Peptide sequences for the liver isoform of Pygl (SEQ ID NO: 1 [human] and 2 [mouse]), which has a monomeric molecular weight of 97 kDa, were observed in the gel band corresponding to approximately 40-50 kDa in serum from the APAP-treated mice only (FIG. 2). Furthermore, different peptides spanning the length of the full protein sequence were detected in this region of the gel. Together, these data indicate that Pygl is cleaved approximately in half and detectable in serum during liver injury. In addition, fragments of peroxisomal acyl-coenzyme A oxidase 1 (Acoxl) and HSD17B4 were also detected in this region of the gel, despite having monomeric molecular weights of >70 kDa. Finally, other fragmented proteins identified in this experiment included lactate dehydrogenase (LDH) and alpha 2-antiplasmin (Serpinf2). Of these fragmented proteins, Pygl produced the strongest signal.The presence of liver injury-induced Pygl fragments in humans was also confirmed using immunoassay methods. (FIG. 3). Western blot analysis was performed on sera from eight patients with liver injury caused by acetaminophen and four healthy control volunteers following depletion of albumin and immunoglobulins.

[0101] The sera from liver injury patients were obtained from the multi-site Acute Liver Failure Study Group (ALFSG). Inclusion criteria for liver injury patients for the ALFSG were altered mentation of any degree (encephalopathy), evidence of moderately severe coagulopathy (INR >1.5), and a presumed acute illness onset of less than 26 weeks overall. Exclusion criteria were cirrhosis, alcohol-induced liver failure, and known pre-existing chronic liver disease. The diagnosis of acetaminophen overdose was made by clinicians at participating ALFSG sites. Sera were depleted of albumin and immunoglobulins using the Pierce Albumin Serum Depletion Kit (ThermoFisher, Waltham, PA; Cat. No. 89875) according to the manufacturer’s instructions. The albumin / immunoglobulin depleted sera were then diluted 1 : 1 with 2x Laemmli Buffer containing a reducing reagent and boiled for 1 minute. The samples were then loaded onto a 4-20% SDS-PAGE gel and proteins in the samples were separated by 2-dimensional electrophoresis. The separated proteins were then transferred to a PVDF membrane for detection. The membrane was blocked with 5% (mass to volume) milk in Tris-buffered saline containing 0.1% Tween 80 for 30 minutes. After blocking, Pygl antibody (PYGL (E4O1P) Rabbit mAb #42103) was diluted 1 : 1,000 in blocking solution, the membrane was immersed in the antibody solution, and the membranesolution mixture was incubated overnight at 4°C. After incubation, the membrane was rinsed with blocking solution without milk to remove the primary antibody. Secondary antibody (HRP-conjugated anti-Rabbit immunoglobulin) was diluted 1 : 10,000 in blocking solution, the membrane was immersed in the secondary antibody solution, and the membrane-solution mixture was incubated for 1 hour in ambient conditions. The membrane was then rinsed again with blocking solution without milk and Pygl was visualized by electrochemiluminescence. The immunoblot contained bands corresponding to intact Pygl and Pygl fragments.

[0102] An ELISA assay was also developed to measure total Pygl in mice and humans with drug-induced liver injury, using two commercially available antibodies that both target the C-terminus of Pygl (FIG. 5). Mice were overdosed with APAP, and serum samples were collected before overdose (0 h) and at 6 and 24 h post-overdose. Human serum samples were analyzed from healthy volunteers (HV) and subjects with acute liver failure (ALF) caused by APAP overdose. The resultsconfirmed that total Pygl levels in serum are increased following APAP overdose, indicating that both the total and fragmented forms of Pygl can serve as specific biomarkers of drug-induced liver injury.

Claims

CLAIMSWe claim:

1. A method of detecting the presence of or increased levels of glycogen phosphorylase (Pygl) in a subject, comprisinga. obtaining a sample from the blood of a subject;b. detecting the presence of or increased levels of Pygl in the sample, wherein the increased levels of Pygl (i) are increased relative to a control and (ii) comprise intact Pygl, fragmented Pygl, and / or total Pygl.

2. The method of claim 1, wherein the presence of or increased levels of Pygl in the blood is associated with liver injury in the subject.

3. The method of claim 1 or 2, wherein the sample is a serum or plasma sample.

4. The method of any one of the preceding claims, wherein at least a portion of the albumin is removed from the sample prior to step (b).

5. The method of any one of the preceding claims, wherein the liver injury is drug-induced liver injury.

6. The method of claim 5, wherein the sample is collected at least six hours after administration of a drug.

7. The method of claim 6, wherein the sample is collected no more than 3 months after administration of a drug.

8. The method of any one of the preceding claims, wherein the method may be repeated at regular intervals during or after administration of a drug.

9. The method of claim 8, wherein the regular intervals are weekly, biweekly or monthly.

10. The method of any one of the preceding claims, wherein the subject is a mammal.

11. The method of claim 10, wherein the mammal is selected from the group consisting of a rodent (mouse or rat), a cat, a dog, a cow, a horse, a pig and a human.

12. The method of any one of the preceding claims, wherein the presence of or increased levels of Pygl is detected using mass spectrometry.

13. The method of any one of the preceding claims, wherein presence of or increased levels of Pygl is detected using an immunoassay.

14. The method of claim 13, wherein the immunoassay is a western blot using an antibody capable of binding to at least a fragmented Pygl.

15. The method of claim 13, wherein the immunoassay is an enzyme-linked immunosorbent assay (ELISA).

16. The method of any one of claims 1-13, wherein the presence of or increased levels of Pygl is detected using a lateral flow assay (LFA).

17. The method of claim 15, wherein step (b) further comprises determining the levels of total Pygl, and wherein the ELISA comprises a capture antibody that binds to both intact and fragmented Pygl and a detection antibody that binds to both intact and fragmented Pygl.

18. The method of claim 15, wherein step (b) further comprises determining the levels of intact Pygl, and wherein the ELISA comprises at least one capture antibody or at least one detection antibody that binds to intact Pygl, but does not bind to fragmented Pygl.

19. The method of claim 15, wherein step (b) further comprises:i) determining the levels of total Pygl in a first ELISA, the ELISA comprising a first capture antibody that binds to both intact and fragmented Pygl and a first detection antibody that binds to both intact and fragmented Pygl;ii) determining the levels of intact Pygl in a second ELISA, the ELISA comprising at least one second capture antibody or at least one second detection antibody that binds to intact Pygl, but does not bind to fragmented Pygl; andiii) quantifying the levels of fragmented Pygl by subtracting the intact Pygl levels from step ii) from the total Pygl levels of i).

20. The method of claim 15, wherein step (b) further comprises determining the levels of total Pygl, and wherein the ELISA comprises a capture antibody and a detection antibody which are both capable of binding to the same portion of Pygl.

21. The method of claim 15, wherein step (b) further comprises determining the levels of intact Pygl, and wherein the ELISA comprises a capture antibody and a detection antibody that do not bind to the same portion of Pygl.

22. The method of claim 15, wherein step (b) further comprises:i) determining the levels of total Pygl in a first ELISA, the ELISA comprising a first capture antibody and a first detection antibody which are both capable of binding to the same portion of Pygl;ii) determining the levels of intact Pygl in a second ELISA, the ELISA comprising a second capture antibody and a second detection antibody that do not bind to the same portion of Pygl; and iii) quantifying the levels of fragmented Pygl by subtracting the intact Pygl levels from step ii) from the total Pygl levels of i).

23. A method of diagnosing liver injury in a subject comprising: detecting presence of or increased levels of fragmented Pygl using the method of any one of the preceding claims, wherein the presence of or increased levels of fragmented Pygl in the blood is indicative of liver injury in the subject.

24. A method of diagnosing liver injury in a subject comprising: detecting presence of or increased levels of intact Pygl using the method of any one of claims 1-22, wherein the presence of or increased levels of intact Pygl in the blood is indicative of liver injury in the subject, and wherein the increased levels of intact Pygl are increased relative to a control.

25. A method of diagnosing liver injury in a subject comprising: detecting presence of or increased levels of total Pygl using the method of any one of claims 1-22, wherein the presence of or increased levels of total Pygl in the blood is indicative of liver injury in the subject, and wherein the increased levels of total Pygl are increased relative to a control.

26. The method of any one of claims 23-25, wherein the liver injury is drug-induced liver injury.

27. The method of any one of claims 23-26, further comprising treating the subject for the liver injury.

28. The method of claim 27, wherein the treatment comprises administering N-acetylcysteine.

29. The method of claim 27, wherein the treatment comprises not administering at least one pharmaceutical that had been administered to the subject prior to the sample being obtained in step (a).

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