WO2024074685A1 - Utilisation de profils d'isoformes d'albumine pour caractérisation d'étiologie et de gravité des lésions hépatiques - Google Patents

Utilisation de profils d'isoformes d'albumine pour caractérisation d'étiologie et de gravité des lésions hépatiques Download PDF

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WO2024074685A1
WO2024074685A1 PCT/EP2023/077721 EP2023077721W WO2024074685A1 WO 2024074685 A1 WO2024074685 A1 WO 2024074685A1 EP 2023077721 W EP2023077721 W EP 2023077721W WO 2024074685 A1 WO2024074685 A1 WO 2024074685A1
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liver
albumin
isoforms
alb
patients
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PCT/EP2023/077721
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Souleiman EL BALKHI
François-Ludovic SAUVAGE
Pierre Marquet
Franck SAINT-MARCOUX
Mohamed-Ali RAHALI
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Institut National de la Santé et de la Recherche Médicale
Centre Hospitalier Universitaire De Limoges
Université De Limoges
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • the present invention is in the field of medicine, in particular hepatology.
  • liver injuries The pathogenesis of liver injuries is complex and involves numerous cellular, molecular, immune and hormonal disturbances (1). This makes the diagnosis of liver diseases a challenging process. The intricacy of these disturbances has led to the examination of a multitude of potential serum biomarkers that reflect underlying disease through cellular pathways, including hepatocellular apoptosis, inflammation or oxidative stress. However, despite the utility of these serum biomarkers (called ‘conventional liver biomarkers’ thereafter) in the assessment of liver injuries and chronic liver diseases, they have limited usefulness in the early stages of liver injuries (2-4).
  • biomarkers such as molecules involved in the fibrosis process including protein-based biomarkers, microRNA or collagens, have been extensively studied. Although some are promising, they have not yet demonstrated the diagnostic or predictive performances required for early dysfunctions or for mid- or long-term liver injuries (5). Therefore, noninvasive (or less invasive), simple, sensitive and specific biomarkers for the early detection of dysfunctions able to lead to liver failure are still needed.
  • HSA human serum albumin
  • HMA Human mercaptalbumin
  • HNA1 Nonmercaptalbumin 1
  • HNA2 Nonmercaptalbumin 2
  • HNA1 plays a pejorative role in decompensated cirrhosis (18) while native HSA has a protective role by reducing the proinflammatory environment present in patients with acutely decompensated cirrhosis (19).
  • HSA has a protective role by reducing the proinflammatory environment present in patients with acutely decompensated cirrhosis (19).
  • Structural alterations involving sites others than Cys34 were reported. Indeed, N- or C-terminal truncated as well as glycated forms were found in plasma samples from patients with acutely decompensated cirrhosis or severe alcoholic hepatitis (9).
  • HSA modifications are directly related to liver injuries and that HSA isoforms are very likely produced because of the chemical environment into the hepatocytes.
  • albumin modifications detected in blood may reflect the dys/function of hepatocytes and represent a versatile tool for the diagnosis and the prognosis of liver injuries and/or diseases.
  • the present invention is defined by the claims.
  • the present invention relates to the use of albumin isoforms profiles for the characterization of the etiology and severity of liver injuries.
  • albumin posttranslational modifications (Alb-PTM) occur very early during the course of liver injuries induced by hepatotoxic substances.
  • native albumin started to decrease in favor of other isoforms 24 hours after the administration of APAP, ethanol or CC14.
  • the nature and the intensity of isoforms were different depending on the hepatotoxic substance.
  • the inventors were able to identify up to 14 albumin isoforms, all of which were also present in control patients.
  • HSA-DA isoform was specific to patients with cirrhosis due to alcohol abuse, HSA+SGGS and HSA+2Glyc were increased specifically in NASH patients, and HSA-DA+Cys with HSA+SO2H were increased only in patients with the mixed form.
  • HSA-DA+Cys with HSA+SO2H were increased only in patients with the mixed form.
  • the term “subject” as used herein refers to any mammal organism.
  • the term subject includes, but is not limited to, humans, nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
  • the term “injury” refers to any damage that directly or indirectly affects the normal functioning.
  • An insult may have a variety of causes including, but not limited to physiological injuries, chemical injuries or physical injuries.
  • the term encompasses acute and chronic injuries.
  • acute injury includes injuries that have recently occurred. For example, an acute injury may have very recently occurred, may have occurred within an hour or less, may have occurred within a day or less, may have occurred within a week or less, or may have occurred within two weeks or less.
  • the term “chronic injury” is an injury that has persisted for a period of time. For example, a chronic injury may have occurred more than two weeks ago, may have occurred more than three weeks ago, may have occurred more than two months ago, or may have occurred more than three months ago.
  • liver injury refers to a state in which the liver function is decreased relative to a normal state. Hepatic dysfunction is characteristic of liver diseases. A number of acute or chronic pathological conditions leads to liver injury. These include, but are not limited to liver abscess, liver cancer, either primary or metastatic, cirrhosis, such as cirrhosis caused by the alcohol consumption or primary biliary cirrhosis, amebic liver abscess, autoimmune hepatitis, biliary atresia, coccidioidomycosis disseminated, portal hypertension hepatic infections (such as hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, or hepatitis E virus), hemochromatosis, hepatocellular carcinoma, pyogenic liver abscess, Reye's syndrome, sclerosing cholangitis, Wilson's disease, drug induced hepatic infections (such as hepati
  • non-alcoholic fatty liver disease has its general meaning in the art and is intended to refer to the spectrum of disorders resulting from an accumulation of fat in liver cells in individuals with no history of excessive alcohol consumption.
  • NAFLD refers to hepatic steatosis.
  • the term NAFLD is also intended to encompass the more severe and advanced form non-alcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma, and virus- induced (e.g., HIV, hepatitis) fatty liver disease.
  • the term "NASH” collectively refers to the state where the liver develops a hepatic disorder (e.g., inflammation, ballooning, fibrosis, cirrhosis, or cancer), or the state where the liver may induce such a pathological condition, and "NASH” is distinguished from "simple steatosis”; i.e., a condition in which fat is simply accumulated in the liver, and which does not progress to another hepatic-disorder-developing condition.
  • a hepatic disorder e.g., inflammation, ballooning, fibrosis, cirrhosis, or cancer
  • cirrhosis refers to a consequence of chronic liver disease characterized by replacement of liver tissue by fibrosis, scar tissue and regenerative nodules (lumps that occur as a result of a process in which damaged tissue is regenerated), leading to loss of liver function.
  • fibrosis characterized by replacement of liver tissue by fibrosis, scar tissue and regenerative nodules (lumps that occur as a result of a process in which damaged tissue is regenerated), leading to loss of liver function.
  • regenerative nodules regenerative nodules
  • the term “etiology” refers to the causes or origins, of diseases or abnormal physiological conditions.
  • severity refers to the degree of symptom intensity experienced, ascertained, formally assessed or reported by a symptomatic subject with a liver injury. Typically, the severity correlates with the Child-Pugh score.
  • the term the “Child-Pugh score” has its general meaning in the art and refers to the score used to assess the prognosis of chronic liver disease, mainly cirrhosis as described by Child CG, Turcotte JG (1964). "Surgery and portal hypertension". In Child CG (ed.). The liver and portal hypertension. Philadelphia: Saunders, pp. 50-64. Although it was originally used to predict mortality during surgery, it is now used to determine the prognosis, as well as the required strength of treatment and the necessity of liver transplantation. The score employs five clinical measures of liver disease including total bilirubin, serum albumin, prothrombin time prolongation (or INR), ascites and hepatic encephalopathy. Each measure is scored 1-3, with 3 indicating most severe derangement. Chronic liver disease is classified into Child-Pugh class A to C, as depicted in Table A.
  • Table A Child-Pugh score and significance.
  • albumin has its general meaning in the art and refers to a globular protein that in humans is encoded by the ALB gene. Serum albumin is the most abundant plasma protein in mammals. Serum albumin is essential for maintaining the oncotic pressure needed for proper distribution of body fluids between intravascular compartments and body tissues. It also acts as a plasma carrier by non-specifically binding several hydrophobic steroid hormones and as a transport protein for hemin and fatty acids. Furthermore, serum albumin has a very long half-life of about 19 days, and its metabolism is well-known. Albumin has also been widely used as a protein stabilizer in commercial pharmaceuticals (Sangastino et al. (2012), Blood, 120(12) 2405-2411).
  • HSA human serum albumin
  • the term “isoform” has its general meaning in the art and refers to the multiple molecular forms of a given protein, and includes proteins differing at the level of (1) primary structure (such as due to alternate RNA splicing, or polymorphisms); (2) secondary structure (such as due to different co- or posttranslational modifications); and/or (3) tertiary or quaternary structure (such as due to different sub-unit interactions, homo- or hetero- oligomeric multimerization).
  • the term “isoform” preferably refers to the multiple molecular forms of a given protein, and includes proteins secondary structure due to different co- or post translational modifications. Said post translational modifications include cysteinylation, homocysteinylation, glutathionylation, glycation, nitrosylation, nitration, oxidation and carbonylation.
  • native albumin refers to the form of albumin that was not subjected to a modification, more particularly a post translational modification.
  • Alb-DA refers to an albumin isoform characterized by a truncation of the N-terminus end.
  • Alb-DA+Cys refers to an albumin isoform characterized by a truncation of the N-terminus end and a cysteinylation of the Cys34.
  • Alb+SO2H refers to an albumin isoform characterized a deoxidation.
  • Alb+SO3H or “Alb-CysO3” refers to an albumin isoform characterized by a trioxidation.
  • Alb+Cys-DHA refers an albumin isoform characterized by a cysteinylation of the Cys34 and a transformation of a free cysteine to a dehydroalanine.
  • Alb+Cys+SNO refers to an albumin isoform characterized by a cysteinylation of the Cys34 and a nitrosylation.
  • Alb+Glyc refers to an albumin isoform characterized by a glycation/
  • Alb+SO2H+Glyc refers to an albumin isoform characterized by a dioxidation and a glycation.
  • Alb+SO3H+Glyc refers to an albumin isoform characterized by a trioxidation and a glycation.
  • Alb+Cys+Glyc-DHA refer to an albumin isoform characterized by a cysteinylation of the Cys34, a glycation and a transformation of a free cysteine to a dehydroalanine.
  • Alb+Cys+Glyc refers to an albumin isoform characterized by a cysteinylation of the Cys34 and a glycation.
  • Alb-SGGS refers to an albumin isoform characterized by a glutathionylation.
  • Alb+2Glyc refers to an albumin isoform characterized by two glycations.
  • Alb+SO3H+2Glyc refers to an albumin isoform characterized by a trioxidation and two glycations.
  • Alb+Cys+2Glyc refers to an albumin isoform characterized by a cysteinylation of the Cys34 and two glycations.
  • the term "profile" means a pattern and relates to the magnitude and direction of change of a number of features.
  • the profile may be interpreted stringently, i.e., where the variation in the magnitude and/or number of features within the profile displaying the characteristic is substantially similar to a reference profile or it may be interpreted less stringently, for example, by requiring a trend rather than an absolute match of all or a subset of feature characteristics.
  • MS mass spectrometry
  • MS refers to an analytical technique to identify compounds by their mass.
  • MS refers to methods of filtering, detecting, and measuring ions based on their m/z.
  • MS technology generally includes (1) ionizing the compounds to form charged species (e.g., ions); and (2) detecting the molecular weight of the ions and calculating their m/z.
  • the compounds may be ionized and detected by any suitable means.
  • a “mass spectrometer” generally includes an ionizer and an ion detector.
  • one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrometric instrument where, due to a combination of magnetic and electric fields, the ions follow a path in space that is dependent upon mass (“m”) and charge (“z”).
  • m mass
  • z charge
  • U.S. Pat. No. 6,204,500 entitled “Mass Spectrometry From Surfaces;”
  • U.S. Pat. No. 6,107,623 entitled “Methods and Apparatus for Tandem Mass Spectrometry;”
  • U.S. Pat. No. 6,268,144 entitled “DNA Diagnostics Based On Mass Spectrometry;” U.S. Pat. No.
  • blood sample means a whole blood, serum, or plasma sample obtained from the patient or the animal.
  • the first object of the present invention relates to a method of determining the etiology and severity of a liver injury in a subject comprising determining the profile of albumin isoforms in a blood sample obtained from the subject wherein the profile indicates the etiology and severity of the liver injury.
  • the method of the present invention comprises the step of detecting a plurality of albumin isoforms. Even, more particularly, the method of the present invention comprises the step of detecting a plurality of albumin isoforms selected from Table 4. In some embodiments, the method of the present invention comprises the step of detecting a plurality of isoforms selected from the group consisting of Alb+SO2H, HSA-CysO3, Alb+Cys-DHA, Alb+Cys, Alb+Cys+SNO, Alb+SO2H+Glyc, Alb+SO3H+Glyc, HAS-SGGS, Alb+2Glyc, Alb+SO3H+2Glyc, and Alb+Cys+2Glyc.
  • the method of the present invention comprises the steps of i) determining the profile of albumin isoforms in the blood sample obtained from the patient, and ii) comparing the profile to one or more reference profiles associated with various liver injuries.
  • the method of the present invention is particularly suitable for detecting any kind of liver injury. In some embodiments, the method of the present invention is particularly suitable for detecting chemical liver injuries. In some embodiments, the method of the present invention is particularly suitable for detecting physical liver injuries. In some embodiments, the method of the present invention is particularly suitable for detecting ischemic liver injuries.
  • the method of the present invention is particularly suitable for the early detection of a liver injury, i.e. the detection of a liver injury before the observation of a symptom.
  • the method of the present invention is particularly suitable for the early detection of early graft dysfunction or non-function in liver transplanted patients.
  • the method of the present invention is particularly suitable for detecting a chemical liver injury induced by a toxicant selected from the group consisting of alcohol, 2,2 Z ,4,4 Z ,5,5 Z -hexachlorobiphenyl (PCB-153), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2-bromoethylamine (BEA), 3-methylcholanthrene, 4-aminophenol (PAP), acetaminophen (APAP), adriamycin, allyl alcohol, amiodarone, amphotericin B, Aroclor 1254, Aroclor 1260, arsenic, aspirin, astemizole, benzene, cadmium, carbamezipine, carbon tetrachloride (CC14), ciprofibrate (cipro), clofibrate, cobalt chloride, corvastatin, cyclosporin A, diethylnitros
  • the method of the present invention is particularly suitable for detecting a liver injury selected from the group consisting of liver abscess, liver cancer, either primary or metastatic, cirrhosis, such as cirrhosis caused by the alcohol consumption or primary biliary cirrhosis, amebic liver abscess, autoimmune hepatitis, biliary atresia, coccidioidomycosis disseminated, portal hypertension hepatic infections (such as hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, or hepatitis E virus), hemochromatosis, hepatocellular carcinoma, pyogenic liver abscess, Reye's syndrome, sclerosing cholangitis, Wilson's disease, drug induced hepatotoxicity, or fulminant or acute liver failure.
  • cirrhosis such as cirrhosis caused by the alcohol consumption or primary biliary
  • the method of the present invention is particularly suitable for detecting a non-alcoholic fatty liver disease, and in particular for detecting NASH.
  • the method of the present invention is particularly suitable for detecting liver fibrosis.
  • the method of the present invention is particularly suitable for detecting cirrhosis.
  • the method of diagnosing described herein is applied to a subject who presents symptoms of liver injury without having undergone the routine screening to rule out all possible causes for liver injury.
  • the methods described herein can be part of the routine set of tests performed on a subject who presents symptoms of liver injury such as jaundice, abdominal pain and swelling, swelling in the legs and ankles, itchy skin, dark urine color, pale stool color, bloody color stool, tar-colored stool, chronic fatigue, nausea or vomiting, loss of appetite, tendency to bruise easily. . .
  • the method of the present invention can be carried out in addition of other diagnostic tools that include ultrasound evaluation (e.g.
  • elastography biopsy and/or quantification of at least one further biomarkers such as levels of blood AST, ALT, ALP, TTT, ZTT, total bilirubin, total protein, albumin, lactate dehydrogenase, choline esterase and the like.
  • a further object to the present invention relates to a method of predicting the worsening of a liver injury comprising the steps of determining the evolution of the profile of albumin isoforms in the blood sample obtained from the patient wherein said evolution predicts the worsening of the liver injury.
  • a further object of the present invention relates to a method of predicting an early allograft liver dysfunction or liver non-function in a liver-transplanted patient comprising the steps of determining the evolution of the profile of albumin isoforms in the blood sample obtained from the patient wherein said evolution predicts the early allograft dysfunction or non-function.
  • the isoforms are detected by mass spectrometry.
  • the isoform is identified visually by its mass shift with respect to native albumin and by taking the maximum intensity in a mass interval of 10 Da around the observed peak of each isoform.
  • Table 4 indicates the different mass shifts that are associated with Alb+SO2H, HSA-CysO3, Alb+Cys-DHA, Alb+Cys, Alb+Cys+SNO, Alb+SO2H+Glyc, Alb+SO3H+Glyc, HSA-SGGS, Alb+2Glyc, Alb+SO3H+2Glyc, and Alb+Cys+2Glyc.
  • Mass spectrometry is performed using a mass spectrometer, which includes an ion source for ionizing the fractionated sample and creating charged molecules for further analysis.
  • Ionization sources used in various MS techniques include, but are not limited to, electron ionization, chemical ionization, electrospray ionization (ESI), photon ionization, atmospheric pressure chemical ionization (APCI), photoionization, atmospheric pressure photoionization (APPI), fast atom bombardment (FAB)/liquid secondary ionization (LSIMS), matrix assisted laser desorption ionization (MALDI), field ionization, field desorption, thermospray/plasmaspray ionization, surface enhanced laser desorption ionization (SELDI), inductively coupled plasma (ICP) and particle beam ionization.
  • ESI electron ionization
  • APCI atmospheric pressure chemical ionization
  • APPI atmospheric pressure photoionization
  • FAB fast atom bombardment
  • LIMS
  • the choice of ionization method may be determined based on the analyte to be measured, type of sample, the type of detector, the choice of positive versus negative mode, etc.
  • the positively charged ions thereby created may be analyzed to determine m/z.
  • Suitable analyzers for determining m/z include quadrupole analyzers, ion trap analyzers, and time-of- flight analyzers.
  • the ions may be detected using one of several detection modes. For example, only selected ions may be detected using a selective ion monitoring mode (SIM), or alternatively, multiple ions may be detected using a scanning mode, e.g., multiple reaction monitoring (MRM) or selected reaction monitoring (SRM).
  • SIM selective ion monitoring mode
  • MRM multiple reaction monitoring
  • SRM selected reaction monitoring
  • a precursor ion also called a parent ion
  • the precursor ion subsequently fragmented to yield one or more fragment ions (also called daughter ions or product ions) that are then analyzed in a second MS procedure.
  • fragment ions also called daughter ions or product ions
  • the MS/MS technique may provide an extremely powerful analytical tool.
  • the combination of filtration/fragmentation may be used to eliminate interfering substances, and may be particularly useful in complex samples, such as biological samples.
  • recent advances in technology such as matrix-assisted laser desorption ionization coupled with time- of-flight analyzers (“MALDI-TOF”) permit the analysis of analytes at femtomole levels in very short ion pulses.
  • MALDI-TOF time- of-flight analyzers
  • MS/MS mass spectrometry steps
  • MS/MS/TOF MS/MS/TOF
  • MALDI/MS/MS/TOF MALDI/MS/MS/TOF
  • SELDI/MS/MS/TOF mass spectrometry SELDI/MS/MS/TOF mass spectrometry.
  • the blood samples are processed to obtain preparations that are suitable for analysis by mass spectrometry.
  • Such purification will usually include chromatography, such as liquid chromatography or capillary electrophoresis, and may also often involve an additional purification procedure that is performed prior to chromatography.
  • chromatography such as liquid chromatography or capillary electrophoresis
  • Various procedures may be used for this purpose depending on the type of sample or the type of chromatography. Examples include filtration, centrifugation, combinations thereof and the like.
  • the pH of the serum sample may then be adjusted.
  • the sample may be purified with a filtration.
  • the filtrate from this filtration can then be purified by liquid chromatography and subsequently subjected to mass spectrometry analysis.
  • HPLC high performance liquid chromatography
  • One or more steps of the methods may be performed using automated machines.
  • one or more purification steps are performed on-line, and more preferably all of the LC purification and mass spectrometry steps may be performed in an on-line fashion.
  • the method of the invention comprises the use of an algorithm. More particularly, the method of the present invention is a computer-implemented method comprising applying, on a set of values for each detected isoforms relative to the subject, a trained model configured to determine the etiology and severity of the liver injury based on the set of values. Typically, the set of values comprises the relative abundance of the different detected isoforms.
  • the model is preliminary trained by supervised learning on a training dataset comprising, for a plurality of individuals of a population, the etiologies and the severities of a liver injury.
  • the method of the invention thus comprises the use of a classification algorithm typically selected from Linear Discriminant Analysis (LDA), Topological Data Analysis (TDA), Neural Networks, Support Vector Machine (SVM) algorithm and Random Forests algorithm (RF).
  • LDA Linear Discriminant Analysis
  • TDA Topological Data Analysis
  • SVM Support Vector Machine
  • RF Random Forests algorithm
  • the term "classification algorithm” has its general meaning in the art and refers to classification and regression tree methods and multivariate classification well known in the art such as described in US 8,126,690; WO2008/156617.
  • the method of the present invention comprises a) detecting the plurality of albumin isoforms; b) implementing a classification algorithm on data relative to the detected isoforms so as to obtain an algorithm output; c) determining the liver injury and severity.
  • the algorithm of the present invention can be performed by one or more programmable processors executing one or more computer programs to perform functions by operating on input data and generating output.
  • the algorithm can also be performed by, and apparatus can also be implemented as, special purpose logic circuitry, e.g., an FPGA (field programmable gate array) or an ASIC (application-specific integrated circuit).
  • processors suitable for the execution of a computer program include, by way of example, both general and special purpose microprocessors, and any one or more processors of any kind of digital computer.
  • a processor will receive instructions and data from a read-only memory or a random access memory or both.
  • the essential elements of a computer are a processor for performing instructions and one or more memory devices for storing instructions and data.
  • a computer will also include, or be operatively coupled to receive data from or transfer data to, or both, one or more mass storage devices for storing data, e.g., magnetic, magneto-optical disks, or optical disks.
  • the algorithm can be implemented in a computing system that includes a back-end component, e.g., as a data server, or that includes a middleware component, e.g., an application server, or that includes a front-end component, e.g., a client computer having a graphical user interface or a Web browser through which a user can interact with an implementation of the invention, or any combination of one or more such back-end, middleware, or front-end components.
  • a back-end component e.g., as a data server
  • a middleware component e.g., an application server
  • a front-end component e.g., a client computer having a graphical user interface or a Web browser through which a user can interact with an implementation of the invention
  • the result given by the methods of the invention may be used as a guide in selecting a therapy or treatment regimen for the subject.
  • the patient can be then eligible for intensive surveillance (e.g., referral to tertiary care centers; intensive control of risk factors), for a selected therapy or transplantation and for inclusion in clinical trials testing new drugs.
  • the treatment consists in any method or drug that could be suitable for the treatment of a liver injury.
  • Some liver problems can be treated with lifestyle modifications, such as stopping alcohol use or losing weight, typically as part of a medical program that includes careful monitoring of liver function.
  • Each liver disease will have its own specific treatment regimen. For example, hepatitis A requires supportive care to maintain hydration while the body's immune system fights and resolves the infection.
  • Patients with gallstones may require surgery to remove the gallbladder.
  • Other diseases may need long-term medical care to control and minimize the consequences of their disease.
  • medications may be required to control the amount of protein absorbed in the diet.
  • Other examples include operations required to treat portal hypertension.
  • the patient can also be eligible for administration of corticosteroids, pentoxifylline, or N-acetylcysteine; antiapoptotics, or vasoactive drugs) or even for liver transplantation.
  • the method of the present invention can also be useful for monitoring the subject.
  • the method of the present invention is also particularly suitable for determining whether a subject suffering from a liver injury achieves a response to a therapy.
  • the method is thus particularly suitable for discriminating responder from non-responder.
  • responder in the context of the present disclosure refers to a subject that will achieve a response, i.e. a subject who is under remission and more particularly a subject who does not suffer from liver injury.
  • a “non-responder” subject includes subjects for whom the disease does not show reduction or improvement after the treatment (e.g. the liver injury remains stable or decreases). For instance, the nature and the abundance of the different isoforms can indeed be monitored during the treatment of the subject and thus can indicate whether the subject achieves a response to the therapy.
  • FIGURES are a diagrammatic representation of FIGURES.
  • FIG. 1 Evolution of albumin isoforms in the different groups of rats exposed to ethanol for different time durations. Native albumin is expressed as percent of the sum of all detected isoforms. Other isoforms are expressed in relative abundance to that of native albumin.
  • FIG. 1 Evolution of albumin isoforms in the different groups of rats exposed to CCI4 for different time durations. Native albumin is expressed as percent of the sum of all detected isoforms. Other isoforms are expressed in relative abundances of the native albumin.
  • Figure 3 Evolution of albumin isoforms in the different groups of rats exposed to APAP at different doses and for different durations. Native albumin is expressed as percent of the sum of all detected isoforms. Other isoforms are expressed in relative abundances of the native albumin.
  • Figure 4 A. median albumin profiles of rats intoxicated with CCh; B. median albumin profiles of rats intoxicated with Ethanol, C. median profiles of rats intoxicated with APAP. The different lines represent the control group for each model; the profiles of groups in which biochemistry tests started to be altered and the groups where albumin profiles were the most disturbed.
  • FIG. 1 PCA-DA plot of the 3 animal models. Each dot represents the whole information of the albumin isoform profile for one rat. The different dots represent rats exposed to ethanol, rats exposed to APAP and are rats exposed to CCh.
  • Figure 6 Histograms of albumin isoforms in cirrhotic patients.
  • FIG. 7 A. PCA-DA plot of the 4 groups of patients. Each dot represents the whole information of the albumin profile for one patient. Square dots are control patients, orange dots represent patients with ALD, circle dots are NASH patients and triangle dots patients with cirrhosis of mixed origin. B. PCA-DA plot of 3 groups of patients: Controls, NASH and mixed origin.
  • Figure 8 A. PCA-DA plot of the 4 groups of patients. Each dot represents the whole information of the albumin profile for one patient. Square dots are control patients, circle dots represent stage A, circles dots stage B, triangle dots represent stage C cirrhosis patients. B. PCA-DA plot of 3 groups of patients: stage A, B and C.
  • EAD+ are patients in whom early allograft dysfunction has been diagnosed based on the LGrAFTIO or the EAD scores.
  • EAD- are patients with no graft dysfunction detected.
  • Each dot represents the whole information of the albumin profile for one patient at VI (24 hours before transplantation) subtracted from the whole information of the albumin profile at V6 (7 days after transplantation).
  • FIG. 10 PLS-DA plot of 2 groups of patients.
  • EAD+ are patients in whom early allograft dysfunction has been diagnosed based on the LGrAFTIO or the EAD scores.
  • EAD- are patients with no graft dysfunction detected.
  • Each dot represents the whole information of the albumin profile for one patient at VI (24 hours before transplantation) subtracted from the whole information of the albumin profile at V3 (72 hours after transplantation).
  • hepatotoxicity was induced by oral administration (gavage) of 2 mL of a 50% EtOH solution prepared in physiological saline (0.9% NaCl) to evaluate the timedependent changes in biochemical markers and histological liver injuries.
  • 6 to 9 rats were followed for 1, 3, 7, 10, or 14 days, respectively. All rats received the daily dose of EtOH.
  • the animals were sacrificed 24 hours after their last intake of EtOH. “Control” rats were followed throughout the duration of the protocol, ie 14 days, and received by gavage only physiological serum (0.9% NaCl).
  • APAP paracetamol
  • Plasma samples were collected in Vacutainer® lithium heparin tube for trace elements (Beckton Dickinson, France) and then centrifuged at 3000 rpm for 10 minutes. The plasma samples were then stored at -80 ° C until analysis. Rat livers were quickly removed and fixed in formalin for histological analysis.
  • livers were cut into sections of 1 to 1.5 cm perpendicular to the major axis to allow homogeneous fixation in a 4% formalin solution for a maximum of 7 days. Samples were stained for light microscopy with hematoxylin and eosin staining and Masson's trichrome stain. The pathologist performed histological analysis blindly, with no knowledge of the different experimental groups.
  • a volume of 20 pL of plasma was diluted with 980 pL of an aqueous 20mM ammonium formate solution with 0.1 % formic acid, and then vortex-mixed. The mixture was centrifuged at 10°C and 14 000 g, and 300 pl of the supernatant was then filtered on a 0.22 pm cellulose acetate filter before injection.
  • Chromatographic separation was performed using a Nexera LC40 system (Shimadzu, noisysiel, France) equipped with a thermostated column compartment and a thermostated autosampler with a six-port switching valve. Samples were analyzed without chromatographic separation under isocratic conditions using a 2mM aqueous ammonium formate solution containing 0.1% formic acid as mobile phase A and a mixture of acetonitrile/mobile phase A (90: 10, by volume) as mobile phase B, programmed as follows: 0-3 min, 50% B.
  • Mass spectrometric detection was performed using a Q-TOF mass spectrometer (TripleTOF® 5600+, Sciex, Concord, Canada) equipped with a DuoSpray ion source and operated in the positive ionization mode.
  • a beta-galactosidase solution was used for internal calibration.
  • the source conditions were as follows: temperature, 200 °C; declustering potential (DP), 250 eV; curtain gas (CUR), 40 units; ion source gas (GS1, GS2), respectively 70 and 10 units; and ionspray voltage floating, 5.5 kV. All MS parameters were controlled by Analyst® TF 1.7 (Sciex). Data were processed with PeakView® 2.2 software (Sciex). m/z ratios were scanned using a TOF MS scan from m/z 900 and 1800 with an accumulation time of 500 ms.
  • the LC-MS data were processed using PeakView® 2.2 software and its Bio Tool Kit 2.2.0 feature (Sciex).
  • the input MS spectra selected from 1300 to 1600 was then deconvolved using a low resolution (5000) from m/z 1000 to 200000.
  • Deconvolution spectra (or profiles) are expressed as intensity versus mass in Da. Estimation of the relative abundance of isoforms.
  • each isoform was determined after calculating the ratio between each centroided peak and the total area represented by the sum of the centroid peaks between 65500 and 67000 for rat serum albumin or 66000 to 67500 for human serum albumin.
  • Each isoform was identified visually by its mass shift with respect to native albumin and by taking the maximum intensity in a mass interval of 10 Da around the observed peak of each isoform.
  • the relative proportion of the intensity of each isoform was calculated by dividing the intensity or area obtained from its deconvoluted spectrum by the summed intensity of all isoforms (between 65500 and 67000) and multiplying it by 100. Data were gathered in an Excel file then analyzed using GraphPad® for the potential isoforms.
  • Relative abundance was calculated as the ratio between the maximum intensity of each identified isoform compared that of the native isoform.
  • the cohort was composed of cirrhotic patients and of patients with no liver dysfunction as controls. Patients were considered as free from liver dysfunction on the basis of their clinical diagnosis and their liver function biochemical tests, namely, aspartate transaminase, alanine transaminase, alkaline phosphatase, y-glutamyltransferase, free and total bilirubin, and albumin levels.
  • Cirrhotic patients were included based on an hepatologist’s diagnosis, their liver function biochemical tests and their Child-Pugh scores.
  • Patient plasma samples were prepared as previously described and albumin isoforms were analyzed and identified as previously described for rat plasma samples.
  • Albumin isoforms were characterized for 38 cirrhotic patients and 52 control patients (with no liver injuries). Among the cirrhotic patients, 18 were diagnosed with alcoholic liver disease (ALD), 5 had a nonalcoholic steatosis (NASH) and 16 were diagnosed with a mixed origin of ALD and NASH. The isoforms detected are depicted in Figure 6 where we can observe a decrease of native albumin in all cirrhotic patients. This decrease was the most important in NASH patients. Generally, the increase of the different isoforms was not homogenous among the three groups of patients (Table 5). HSA-DA+Cys and HSA+SO2H were only significantly increased in patients with cirrhosis of mixed origin.
  • HSA+SGGS and HSA+2Glyc were increased only in NASH patients.
  • PCA- DA showed a clear clustering between the 4 groups, namely, control patients, NASH patients, ALD patients and the mixed-cirrhosis patients (Figure 7).
  • stage A (calculated on the basis of Child- Pugh score)
  • stage B (calculated on the basis of Child- Pugh score)
  • stage C (calculated on the basis of Child- Pugh score)
  • PCA-DA showed a clear clustering of the different stages of cirrhosis and the control patients (Figure 8).
  • Albumin isoforms were characterized in 38 liver-transplanted patients at different times: VI (24h before the transplantation), V2 (during the transplantation), V3 (24h after the transplantation), V4 (48h after the transplantation), V5 (72h after the transplantation), V6 (7 days after the transplantation).
  • VI 24h before the transplantation
  • V2 disuring the transplantation
  • V3 24h after the transplantation
  • V4 48h after the transplantation
  • V5 72h after the transplantation
  • V6 7 days after the transplantation.
  • albumin isoforms profiles when taken individually at each time, were not able to discriminate patients with an early allograft dysfunction, the subtraction of the profiles obtained at V6 -VI or V6-V3 allowed a clear clustering of these patients as shown in figure 9 and figure 10 respectively. Discussion:
  • albumin posttranslational modifications (Alb-PTM) occur very early during the course of liver injuries induced by hepatotoxic substances.
  • native albumin started to decrease in favor of other isoforms 24 hours after the administration of APAP, ethanol or CCh.
  • the nature and the intensity of isoforms were different depending on the hepatotoxic substance.
  • a tertiary pathway for the oxidation of ethanol involves catalase, a peroxisomal enzyme that also catalyses the removal of reactive oxygen species (e.g. H2O2).
  • Acute exposure to EtOH leads to an adaptive increase in EtOH metabolism within 2 to 3 hours, in both rodent and human livers. This involves the so-called swift increase in alcohol metabolism (SIAM), defined experimentally as a rapid increase in hepatic alcohol metabolism and mitochondrial respiration after a single high dose of alcohol.
  • SIAM swift increase in alcohol metabolism
  • High demand for O2 during SIAM leads to zones of hypoxia, especially in pericentral (centrilobular) regions of liver lobules, which may contribute to liver injury.
  • the very rapid posttranslational modifications of albumin in our EtOH model are consistent with the rapid onset liver injury described here.
  • EtOH changes the gut microbiome, causing bacterial overgrowth and increasing formation of toxic/proinflammatory products. EtOH consumption also promotes hepatic ROS and RNS formation.
  • EtOH increases CYP2E1, largely by a posttranscriptional mechanism involving stabilization against proteolysis. CYP2E1 generates superoxide (O2"), which then forms highly reactive peroxynitrite (ONOO-) by reaction with NO, and hydroxyl radical ( OH) by the Fenton reaction. In the presence of EtOH, the 1 -hydroxy ethyl radical is also formed. Therefore, ROS, RNS and other radical species increase after EtOH ingestion. These radicals attack and damage proteins, lipids, and DNA, induce mitochondrial permeability transition (MPT), cause cell death, and trigger inflammatory processes.
  • MPT mitochondrial permeability transition
  • albumin is the most abundant protein produced in hepatocytes and due to its great reactivity, it is naturally the most prone to posttranslational modifications due to the abovedescribed reactions, which leads to the increase of albumin isoforms as observed in our ethanol model. However, it is still difficult to explain the increase of each isoform individually, since the chemical environment and the reactions at stake are not yet fully understood.
  • CCh is metabolized in the liver by CYP2E1 and CYP2B1 enzymes and is converted into a highly reactive trichloromethyl (CCh-) radical, ultimately leading to hepatotoxic damage, inflammation and fibrosis within 6 to 12 weeks.
  • CCh- highly reactive trichloromethyl
  • AST and ALT were significantly increased as soon as DI, and BILID after 7 days.
  • the CCh- radical produced after exposure to CCh, can bind to cellular molecules (nucleic acid, protein, lipid), impairing crucial cellular processes such as lipid metabolism, with the potential outcome of fatty degeneration leading to steatosis.
  • CCh- reacts with oxygen to form the trichloromethylperoxy radical CChOO-, another highly reactive species.
  • CChOO- initiates a chain reaction of lipid peroxidation, which attacks and destroys polyunsaturated fatty acids, in particular those associated with phospholipids. These affect the permeability of mitochondrial, endoplasmic reticulum, and plasma membranes.
  • CCh activates tumor necrosis factor (TNF)alpha, nitric oxide (NO), and transforming growth factors (TGF)-alpha and -beta in the cell, processes that appear to direct the cell primarily toward (self-)destruction or fibrosis (23).
  • TNF tumor necrosis factor
  • NO nitric oxide
  • TGF transforming growth factors
  • NAPQI -acetyl-p-benzoquinone imine
  • nitric oxide within the mitochondria to produce highly reactive peroxynitrite, which nitrates mitochondrial proteins such as manganese superoxide dismutase (MnSOD).
  • MnSOD manganese superoxide dismutase
  • ASK1 apoptosis signal-regulating kinase 1
  • ASK1 along with activated mixed-lineage kinase 3 (MLK3) then activated c-jun N-terminal kinase (JNK) to its phosphorylated form through MKK4 phosphorylation.
  • JNK c-jun N-terminal kinase
  • Phosphorylated JNK translocates to the mitochondria and binds to Sab on the outer mitochondrial membrane, which, through a Src-mediated pathway, further inhibits mitochondrial electron transport. This amplifies mitochondrial oxidant stress, which is further exacerbated by translocation of Bax and glycogen synthase kinase-3p (GSK-3P) from the cytosol to the mitochondria.
  • GSK-3P glycogen synthase kinase-3p
  • DAMPs bind to pattern recognition receptors such as toll-like receptors (TLRs) on inflammatory cells and transcriptionally activate cytokine formation in inflammatory cells (25).
  • TLRs toll-like receptors
  • APAP hepatotoxicity is a time-dependent event involving a number of different phases critical for the injury and recovery process: i) the metabolism phase that includes NAPQI production and glutathione depletion occurs within 0 to 3 hours after APAP administration; ii) the early injury phase that includes JNK activation and mitochondrial translocation, mitochondrial BAX translocation, mitochondrial superoxide formation, MPT, glutathione recovery and ALT/AST increase in plasma occurs within 2 to 6 h; iii) the late injury/early recovery phase that includes ALT/AST increase in plasma, necrosis, innate immune response occurs within 12 to 24h; and finally iv) the regeneration phase where resolution of necrosis could be encountered occurs within 24 to 96 h (26).
  • necrosis was mainly observed in rats given 4 g/kg APAP, and inflammation was noted for all doses.
  • Native albumin in this model did not decrease 24 hours post APAP and was even barely detectable at 72h , which is consistent with the mechanism of APAP toxicity described here. The sharp decrease of native albumin was associated with an increase in other isoforms. Among them, Alb+SGGS was specific to this APAP model. As we did not observe any normalization of native albumin, we can assume that the regeneration phase was not reached in our experimental conditions.
  • albumin can undergo several chemical modifications on several sites of its amino acids chain, including: cysteinylation, homocysteinylation, glutathionylation, glycation, nitrosylation, nitration, oxidation and carbonylation.
  • cysteinylation homocysteinylation
  • glutathionylation glutathionylation
  • glycation nitrosylation
  • nitration oxidation and carbonylation
  • the deconvoluted MS spectrum is capable of identifying unknown modifications because the addition or subtraction of a structural modification result in a characteristic mass shift, equal to the mass of the specific modification (for example, a mass difference of 163 Da for a glycation).
  • the 17 isoforms identified had to be “visually detectable” to be included, increasing the risk of leaving behind minor isoforms with low signals. Therefore, to investigate the differences in isoform distribution among the 3 animal models, we decided to integrate the whole information from each spectrum in PCA-DA. The profiles obtained with the 3 models were nicely separated ( Figure 5), suggesting that the Alb profile of each model depends on the different chemical environment and mechanisms of intracellular reactions generated by each substance, as previously explained.
  • albumin isoform profiles represent rich data that could help diagnosing liver diseases, staging them and sorting out their origin.
  • D2g-Dl is the group of rats that were sacrificed 24h after receiving a dose of 2 g/kg of APAP.
  • D2g-D3 is the group of rats that were sacrificed 72h after receiving a dose of a dose of 2g/kg.
  • D3g-Dl is the group of rats that were sacrificed 24h after receiving 3g/kg of APAP.

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Abstract

Les inventeurs ont supposé que chaque type de lésion hépatique peut être révélé par un profil spécifique de modifications post-traductionnelles de la sérumalbumine humaine (HSA). Par conséquent, l'objectif des inventeurs a été d'étudier le motif d'isoformes d'albumine chez des rats intoxiqués avec de l'acétaminophène (APAP), de l'éthanol et du CCI4. Le second objectif était d'explorer le potentiel de ces isoformes en tant que biomarqueurs de lésions spécifiques du foie. Les résultats démontrent que les modifications post-traductionnelles de l'albumine (Alb-PTM) interviennent très tôt au cours des lésions hépatiques induites par des substances hépatotoxiques. Dans trois modèles animaux, l'albumine native a commencé à diminuer au profit d'autres isoformes 24 heures après l'administration d'APAP, d'éthanol ou de CCI4. Il est intéressant de noter que la nature et l'intensité des isoformes sont différentes en fonction de la substance hépatotoxique. Dans une cohorte de patients cirrhotiques, les inventeurs ont pu identifier jusqu'à 14 isoformes d'albumine, tous également présents chez les patients témoins. Cependant, les inventeurs ont observé que l'augmentation de l'isoforme HSA-DA était spécifique aux patients atteints d'une cirrhose due à l'abus d'alcool, que les isoformes HSA+SGGS et HSA+2Glyc étaient augmentés spécifiquement chez les patients avec stéatohépatite non alcoolique, et que les isoformes HSA-DA+Cys et HSA+SO2H n'étaient augmentés que chez les patients atteints de la forme mixte. En outre, aucun isoforme spécifique n'a été observé, permettant de discriminer clairement les différents stades de la maladie hépatique, mais l'analyse en composantes principales de l'ensemble des données SM a permis de séparer parfaitement les patients atteints de cirrhose avec différents scores de Child-Pugh et les patients témoins. La présente invention concerne donc l'utilisation des profils d'isoformes d'albumine pour la caractérisation de l'étiologie et de la gravité des lésions hépatiques.
PCT/EP2023/077721 2022-10-07 2023-10-06 Utilisation de profils d'isoformes d'albumine pour caractérisation d'étiologie et de gravité des lésions hépatiques WO2024074685A1 (fr)

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