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WO2013049509A1 - Panel de dosage pour la stéatohépatite non alcoolique - Google Patents

Panel de dosage pour la stéatohépatite non alcoolique Download PDF

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Publication number
WO2013049509A1
WO2013049509A1 PCT/US2012/057826 US2012057826W WO2013049509A1 WO 2013049509 A1 WO2013049509 A1 WO 2013049509A1 US 2012057826 W US2012057826 W US 2012057826W WO 2013049509 A1 WO2013049509 A1 WO 2013049509A1
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Prior art keywords
biomarkers
keratin
subject
nash
igfbp
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Stephen H CALDWELL
James T. PATRIE
Curtis K. ARGO
Reid W. Von Borstel
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Defined Diagnostics LLC
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Wellstat Diagnostics LLC
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Priority to AU2012315784A priority Critical patent/AU2012315784A1/en
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4742—Keratin; Cytokeratin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4745—Insulin-like growth factor binding protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91188—Transferases (2.) transferring nitrogenous groups (2.6)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis
    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin

Definitions

  • the present disclosure relates to methods of detecting and monitoring non-alcoholic fatty liver disease (NAFLD) in a subject.
  • NAFLD non-alcoholic fatty liver disease
  • the present disclosure relates to methods of screening for non-alcoholic steatohepatitis (NASH) and monitoring the effect of therapy on a subject with NASH.
  • NASH non-alcoholic steatohepatitis
  • NAFLD nonalcoholic fatty liver disease
  • NASH nonalcoholic steatohepatitis
  • liver biopsy is the current gold standard for diagnosing NASH, however, biopsy may be costly, invasive and subjective.
  • a biomarker or set of biomarkers in blood that correlate with liver histology may identify patients who have no need for a biopsy, and also provide early detection of NASH, as well as information about disease progression or regression over time, which may improve patient treatment and outcome.
  • Nonalcoholic fatty liver disease is the most common form of chronic liver disease in both children and adults. It encompasses a wide spectrum of conditions associated with overaccumulation of fat in the liver ranging from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. Simple steatosis is the most common form of NAFLD and typically follows a benign nonprogressive clinical course. In contrast, NASH is a potentially serious condition, since as many as 25% of these patients may progress to cirrhosis and experience complications of portal hypertension, liver failure, and hepatocellulate carcinoma.
  • NASH nonalcoholic steatohepatitis
  • heptocyte apoptosis a specific form of cell death, may play an important role in liver injury and disease progression in NAFLD. It has been shown that caspase activation and liver cell apoptosis are prominent pathological features of human NAFLD. Moreover, the degree of apoptosis correlated with the severity of steatohepatitis and the stage of fibrosis. (Feldstein, A. et al., "Cytokeratin-18 Fragment levels as noninvasive biomarkers for nonalcoholic steatohepatitis: A Multicenter Validation Study," Am. Assoc. Study Liver Diseases, Wiley InterScience, 2009.)
  • the apoptotic pathway is composed of two arms: the intrinsic pathway (initiated by cellular stress) and extrinsic pathway (stimulated through a death receptor-mediated process). Both pathways are suspected to be involved in the pathogenesis of NASH.
  • the effector caspases in particular caspase-3 and caspase-7 are activated.
  • These specific intracellular proteases are known to cleave several cellular substrates, including keratin-18 (K- 18), the major intermediate filament protein in the liver.
  • K- 18 keratin-18
  • Antibodies against caspase- generated K-18 fragments have been shown to specifically label early apoptotic cells.
  • liver biopsy may be recommended for more definitive assessment despite presence of risk and lack of a standard follow-up interval.
  • markers of cell injury such as keratin 18 fragments and markers of insulin metabolism correlate to histological severity in NASH (Younossi et al., Obes. Surg. 2010), but their utility in monitoring therapy is uncertain.
  • the present disclosure generally provides methods of screening a subject to predict the likelihood of significantly active histological nonalcoholic steatohepatitis (NASH) in a subject, comprising measuring the level of at least four biomarkers selected from the group consisting of adiponectin (ADPN), full- length keratin-18 (K-18), caspase-3-cleaved keratin-18 (C3C K-18), insulin-like growth factor binding protein 1 (IGFBP-1 ), and alanine aminotransferase (ALT), in a sample derived from the subject, and analyzing the level of the at least four biomarkers against reference values for those biomarkers.
  • ADPN adiponectin
  • K-18 full- length keratin-18
  • C3C K-18 caspase-3-cleaved keratin-18
  • IGFBP-1 insulin-like growth factor binding protein 1
  • ALT alanine aminotransferase
  • NASH non-alcoholic steatohepatitis
  • methods of screening a subject to identify the presence or absence of non-alcoholic steatohepatitis (NASH) in a subject comprising measuring the level of at least four biomarkers selected from the group consisting of adiponectin (ADPN), full-length keratin-18 (K-18), caspase-3- cleaved keratin-18 (C3C K-18), insulin-like growth factor binding protein 1 (IGFBP-1 ), and alanine aminotransferase (ALT), in a sample derived from the subject, and analyzing the level of each of the at least four biomarkers against reference values for those biomarkers.
  • ADPN adiponectin
  • K-18 full-length keratin-18
  • C3C K-18 caspase-3- cleaved keratin-18
  • IGFBP-1 insulin-like growth factor binding protein 1
  • ALT alanine aminotransferase
  • the present disclosure further relates to methods of measuring the progression of non-alcoholic fatty liver disease (NAFLD) to non-alcoholic NAFLD.
  • NAFLD non-alcoholic fatty liver disease
  • NASH steatohepatitis
  • NASH steatohepatitis
  • ADPN adiponectin
  • K-18 full- length keratin-18
  • C3C K-18 caspase-3-cleaved keratin-18
  • CRP C-Reactive Protein
  • IGFBP-1 insulin-like growth factor binding protein 1
  • ALT alanine aminotransferase
  • the present disclosure relates to methods of measuring the efficacy of a therapy for treatment of NASH in a subject by measuring the level of at least four biomarkers selected from the group consisting of adiponectin (ADPN), full- length keratin-18 (K-18), caspase-3-cleaved keratin-18 (C3C K-18), C-Reactive Protein (CRP), insulin-like growth factor binding protein 1 (IGFBP-1 ), and alanine aminotransferase (ALT), in samples derived from the subject before and after the subject undergoes the therapy, and analyzing the level of the biomarkers after therapy against the values for those biomarkers before therapy.
  • ADPN adiponectin
  • K-18 full- length keratin-18
  • C3C K-18 caspase-3-cleaved keratin-18
  • CRP C-Reactive Protein
  • IGFBP-1 insulin-like growth factor binding protein 1
  • ALT alanine aminotransferase
  • the present disclosure is also directed to a kit comprising a package, the package containing at least four agents for measuring the level of at least four biomarkers of interest, wherein the at least four biomarkers are selected from the group consisting of adiponectin (ADPN), full-length keratin-18 (K-18), caspase-3-cleaved keratin-18 (C3C K-18), C-Reactive Protein (CRP), insulin-like growth factor binding protein 1 (IGFBP-1 ), and alanine aminotransferase (ALT).
  • ADPN adiponectin
  • K-18 full-length keratin-18
  • C3C K-18 caspase-3-cleaved keratin-18
  • CRP C-Reactive Protein
  • IGFBP-1 insulin-like growth factor binding protein 1
  • ALT alanine aminotransferase
  • the present disclosure is based on the finding of a correlation between histological activity (NAS value) or change in NAS score, in patients with NASH and the biomarkers adiponectin (ADPN), full-length keratin-18 (K-18), caspase-3-cleaved keratin-18 (C3C K-18), C-Reactive Protein (CRP), insulin-like growth factor binding protein 1 (IGFBP-1 ), and alanine aminotransferase (ALT), as described in the examples.
  • ADPN histological activity
  • K-18 full-length keratin-18
  • C3C K-18 caspase-3-cleaved keratin-18
  • CRP C-Reactive Protein
  • IGFBP-1 insulin-like growth factor binding protein 1
  • ALT alanine aminotransferase
  • This finding provides a predictive model for NAS values, which can be used for effective, non-invasive diagnosis of NASH as well as non-invasive assessment of NASH progression or the outcomes of therapeutic intervention, especially for short-term interval evaluations where re-biopsying is impractical.
  • FIG. 1 is a collection of box and whisker plots illustrating the empirical distributions of the potential predictors of NASH status of tested subjects (NAS ⁇ 4, NAS ⁇ 4);
  • FIG. 2 is a graphical illustration depicting the relationship between the value of the predictor variable and the predicted probability of a NAS ⁇ 4 value of a subject.
  • FIG. 3 is a graphical illustration depicting the ranking of the predictors of NASH status (NAS ⁇ 4, NAS ⁇ 4) when the ranking criterion is based on the difference between the observed Wald chi-squared statistic and the expected Wald chi-squared statistic under the null hypothesis of no partial association.
  • the present disclosure relates to methods of detecting and monitoring non-alcoholic fatty liver disease (NAFLD) in a subject.
  • the present disclosure also relates to methods of screening for non-alcoholic steatohepatitis (NASH) and monitoring the effect of therapy on a subject with NASH.
  • a method for screening a subject to predict the likelihood of significantly active histological non-alcoholic steatohepatitis (NASH) in a subject can comprise the step of measuring the levels of at least four biomarkers in a sample derived from the subject.
  • the method can further comprise analyzing the levels of the at least four biomarkers against references values for those biomarkers.
  • a method for screening a subject to identify the presence or absence of non-alcoholic steatohepatitis (NASH) in a subject can comprise measuring the level of at least four biomarkers in a sample derived from the subject. The method can further comprise analyzing the levels of the at least four biomarkers against reference values for those biomarkers.
  • NASH non-alcoholic steatohepatitis
  • biomarkers for screening a subject to predict the likelihood of significantly active histological non-alcoholic steatohepatitis (NASH) and/or to identify the presence or absence of NASH in a subject include, but are not limited to, adiponectin (ADPN), full-length keratin-18 (K-18), caspase-3-cleaved keratin-18 (C3C K-18), and insulin-like growth factor binding protein 1 (IGFBP-1 ).
  • Adiponectin and insulin-like growth factor binding protein 1 (IGFBP-1 ) are naturally found in the body of a subject and can be obtained from numerous commercial vendors of antibodies or ELISA kits.
  • the antibodies for full-length keratin-18 are monoclonal antibodies, such as, for example, the M6 and M5 Keratin 18 antibodies (PEVIVA AB).
  • the antibodies for caspase-3-cleaved keratin-18 are monoclonal antibodies, such as, for example, the M30 ® antibody (PEVIVA AB).
  • the M30 Apoptosense ® ELISA and M65 ® ELISA kits, both manufactured by PEVIVA AB were used in some of the experiments related to the caspase-3-cleaved K18 fragments and the full-length K-18, respectively.
  • the number of biomarkers measured can also be at least one or at least two or at least three biomarkers. It is further contemplated that additional or alternative biomarkers can be used in addition to or as a substitution for one or more of the biomarkers listed herein.
  • alternative suitable biomarkers include, but are not limited to, resistin, insulin, fetuin, tumor necrosis factor-alpha (TNF-a), interleukin 6 (IL-6), or interleukin 8 (IL-8), C-Reactive Protein (CRP), alanine aminotransferase (ALT), or any combination thereof.
  • NAFLD non-alcoholic fatty liver disease
  • NASH steatohepatitis
  • monitoring the improvement of NASH-like conditions to NAFLD-like conditions over time in a subject can comprise measuring the level of at least four biomarkers in a sample derived from the subject at two or more time points.
  • the method can further comprise analyzing the level of the at least four biomarkers at a second or later point in time against the values for those biomarkers at an earlier point in time.
  • a method for monitoring the efficacy of a therapy for treatment of non-alcoholic steatohepatitis (NASH) in a subject can comprise measuring the level of at least four biomarkers in a sample derived from the subject before and after the subject undergoes the therapy. The method can further comprise analyzing the level of the at least four biomarkers after therapy against the reference values for those biomarkers before therapy.
  • NASH non-alcoholic steatohepatitis
  • biomarkers for monitoring the progression of NAFLD to NASH or the improvement of NASH-like conditions to NAFLD-like conditions over time and/or monitoring the efficacy of therapy in a subject include, but are not limited to, adiponectin (ADPN), full-length keratin-18 (K-18), caspase-3- cleaved keratin-18 (C3C K-18), C-Reactive Protein (CRP), insulin-like growth factor binding protein 1 (IGFBP-1 ), and alanine aminotransferase (ALT).
  • ADPN, CRP, IGFBP-1 , and ALT are naturally found in the body of a subject and can also be obtained from various commercial sources.
  • the number of biomarkers measured can also be at least one or at least two or at least three biomarkers. It is further contemplated that additional or alternative biomarkers can be used in addition to or as a substitution for one or more of the biomarkers listed herein.
  • measuring the level of the biomarkers can comprise the steps of reacting with a specific antibody against the biomarkers, or against any fragment of a biomarker containing an antigenic determinant.
  • the antibody can comprise, for example, a whole immunoglobulin molecule, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a recombinant fragment of antibody, an Fab fragment, an Fab' fragment, an F(ab')2 fragment, an Fv fragment, an scFv fragment, or any combination thereof.
  • samples from a subject can be analyzed.
  • suitable samples include, but are not limited to, serum, blood, plasma, urine, saliva, cerebrospinal fluid, tissue, or a tissue extract.
  • the samples can be diluted prior to analysis.
  • the levels of the biomarkers within the samples can be measured using any conventional quantitative or qualitative assay technique useful for measuring the presence of a biomarker such as competitive and non-competitive immunoassay formats, antigen capture assays, and two-sandwich assays.
  • acceptable assay methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunofluorescent assay (IFA), chemiluminescent assay, electrochemiluminescent (ECL) assay, sandwich assay, capillary electrophoresis- based immunoassay (CEIA), magnetic capture, microsphere capture, and/or western blotting.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • IFA immunofluorescent assay
  • ECL electrochemiluminescent
  • CEIA capillary electrophoresis- based immunoassay
  • the levels of the various biomarkers can be compared to their respective reference values using any conventional technique, for example, ELISA, immunofluorescent assay (IFA), and chemiluminescent assay. Additionally, it has been found that multivariable logistic regression analysis is a powerful tool for analyzing biomarker levels for use in the methods disclosed herein.
  • the reference value ranges for each of the biomarkers can represent, for example, a subject without NASH or a subject without NAFLD.
  • the analysis can calculate, for example, an optimal threshold for detecting NASH.
  • a threshold value can be selected to provide a sensitivity and/or a specificity for detecting NASH in a certain range.
  • the methods can further comprise analyzing the Body Mass Index (BMI) of the subject.
  • BMI Body Mass Index
  • the methods of the present disclosure are suitable for any mammalian subject, such as a human subject.
  • non-alcoholic fatty liver disease or "NAFLD”
  • NAFLD non-alcoholic steatohepatitis
  • SMSH non-alcoholic steatohepatitis
  • simple steatosis refers to the definitions as currently used and accepted in the scientific community.
  • simple steatosis is the most common form of NAFLD and typically follows a benign nonprogressive clinical course.
  • the patients exhibit an accumulation of fat in the liver cells without significant inflammation and
  • the patients with NASH exhibit inflammation and hepatocellular damage and sometimes fibrosis, which may then progress to cirrhosis.
  • the NASH patients may experience complications of portal hypertension, liver failure, and hepatocellular carcinoma.
  • NASH non-alcoholic steatohepatitis
  • NAS non-alcoholic fatty liver disease Activity Score
  • the total NAS value represents the sum of scores for steatosis, lobular inflammation, and ballooning, and ranges from 0-8, as presented in Table 1 , depicting NAFLD Activity Score (NAS) and Fibrosis Staging, which is widely accepted and well-known in the art.
  • Diagnosis of NASH should be made first, then NAS is used to grade activity.
  • NAS values of 0-2 occurred in cases largely considered not diagnostic of NASH, values of 3-4 were evenly divided among those considered not diagnostic, borderline, or positive for NASH. Values of 5-8 occurred in cases that were largely considered diagnostic of NASH.
  • This category is included to accommodate cases with
  • NASH-like conditions means a condition featuring both fatty liver and inflammation, regardless of etiology. For example, many of the histological signs of NASH can occur in patients with hepatitis C; the assay panel of this disclosure is applicable to detection and monitoring of liver damage in hepatitis C and other conditions overlapping with NASH pathology, including but not limited to liver damage caused by toxic chemicals, infections, autoimmune and autoinflammatory diseases.
  • NAFLD-like conditions as used herein means liver conditions featuring fatty liver with little or no inflammatory infiltrate or fibrosis, but with variable degrees of hepatic insulin resistance, whether caused by infection, chemical toxins or autoimmune or autoinflammatory processes.
  • kits for use in the methods of the present teachings are provided.
  • a kit can comprise a package containing at least one agent for measuring the levels of selected biomarkers, such as adiponectin (ADPN), full-length keratin-18 (K-18), caspase-3-cleaved keratin-18 (C3C K-18), and insulin-like growth factor binding protein 1 (IGFBP-1 ).
  • ADPN adiponectin
  • K-18 full-length keratin-18
  • C3C K-18 caspase-3-cleaved keratin-18
  • IGFBP-1 insulin-like growth factor binding protein 1
  • the package can contain at least two agents, at least three agents, at least four agents, or more than four agents depending on the number and kinds of biomarkers being measured.
  • the package can contain at least one agent for measuring the levels of selected biomarkers, such as full-length keratin-18 (K-18), caspase-3-cleaved keratin-18 (C3C K-18), C-Reactive Protein (CRP), insulinlike growth factor binding protein 1 (IGFBP-1 ), and alanine aminotransferase (ALT). It is contemplated that the package can contain at least two agents, at least three agents, at least four agents, or more than four agents depending on the number and kinds of biomarkers being measured.
  • selected biomarkers such as full-length keratin-18 (K-18), caspase-3-cleaved keratin-18 (C3C K-18), C-Reactive Protein (CRP), insulinlike growth factor binding protein 1 (IGFBP-1 ), and alanine aminotransferase (ALT). It is contemplated that the package can contain at least two agents, at least three agents, at least four agents, or more than four agents depending on the number and kinds of biomarkers being measured.
  • the agents can comprise at least one of an antibody of adiponectin (ADPN), an antibody of full-length keratin- 18 (K-18), an antibody of caspase-3-cleaved keratin-18 (C3C K-18), an antibody of C-Reactive Protein (CRP), an antibody of insulin-like growth factor binding protein 1 (IGFBP-1 ), and an antibody of alanine aminotransferase (ALT).
  • ADPN adiponectin
  • K-18 full-length keratin- 18
  • C3C K-18 caspase-3-cleaved keratin-18
  • CRP C-Reactive Protein
  • IGFBP-1 insulin-like growth factor binding protein 1
  • ALT alanine aminotransferase
  • all of the measuring agents in the kit can be antibodies of the respective biomarkers to be detected.
  • the antibodies for full-length keratin-18 are monoclonal antibodies, such as, for example, the M6 and M5 Keratin 18 antibodies (PEVIVA AB).
  • the antibodies for caspase-3-cleaved keratin-18 are monoclonal antibodies, such as, for example, the M30 ® antibody (PEVIVA AB).
  • the kit may further comprise one or more control reference samples. It is further contemplated that the kit can contain a package with individual containers for each of the antibodies.
  • Instructions for using the kit may be provided in the package or separately.
  • the instructions can comprise explanations on using the contents of the kit and on measuring the levels of the biomarkers.
  • the instructions may also include reference levels for comparing to the detected levels of the biomarkers.
  • Anti-HAMA Antibody Diluent (100 mM sodium phosphate, 150 mM NaCI, 0.5% BSA, 0.5% B IgG, 0.1 % Brij 35, 0.1 % MIT, 50 g/mL MAK33-lgG1/lgG Poly, 25 g/mL HBR); (2) Conjugate Diluent, aka Conjugate Buffer.
  • NAFLD/NASH patient serum samples were collected (from S. Caldwell, MD at University of Virginia) with biopsies beginning at one year from a diagnosis of NAFLD. Thirty-three of those patients with clinically diagnosed fatty liver disease had two liver biopsies performed. One biopsy was performed prior to an intervention (placebo, omega 3 fatty acid), while the other biopsy was performed post-intervention. Each liver biopsy was evaluated by a professional hepatologist who was blind to the intervention and the biopsy was given scores for fat, inflammation, and ballooning, and similarly scored by a professional liver histopathologist, also blinded to the intervention. Based on these scores, the biopsy was then assigned a NAFLD Activity Scores (NAS). Thirteen patients had a histological "response" to NAS score ⁇ 4.
  • each patient had two blood-draws. One draw was taken before the intervention and one draw was taken post-intervention. Fasting blood samples at study entry and follow-up were coded for blinded testing of adiponectin (ADPN), caspase-3-cleaved keratin-18 (C3C K-18), full-length keratin-18 (K-18), C- Reactive Protein (CRP), insulin-like growth factor binding protein 1 (IGFBP-1 ), and alanine aminotransferase (ALT). (ALT was assayed for using the Architect Chemical Analyzer (Abbott). The reaction detects conversion of alanine to pyruvate and then lactate.) The patient data are summarized in Tables 2A-2C.
  • Table 2A - Combined Assay Results Pre-lntervention provides the combined results generated from each of the assays completed (ADPN, C3C K-18, K-18, IGFBP-1 , CRP and ALT), and includes the initial NAS value for each patient clinically diagnosed with fatty liver disease tested prior to the intervention.
  • Table 2B - Combined Assay Results Post- Intervention with Response provides data generated from the tested patients that exhibited a change in NAS >2 after the intervention resulting in an average NAS value below 4, regardless of the starting NAS value.
  • Table 2C - Combined Assay Results Post-Intervention with Non-Response provides data generated from those patients that exhibited a change in NAS ⁇ 2. While many of these patients did actually have a response to the intervention, for purposes of the study, the change in the NAS value ⁇ 2 did not drop the average NAS value below 4, thereby indicating that these patients still were affected with NASH following the intervention.
  • Table 3 provides data generated from twenty randomly selected normal human serum samples received from BioReclamation and includes reference ranges for some of the assays completed (ADPN, IGFBP-1 , CRP).
  • Adiponectin in the normal human serum samples were quantitated using an eight-point calibrator curve with an analytical range from 0 - 100 ng/mL, and quality controls (0.18, 1 .8, and 18 ⁇ g/mL) diluted in DILUENT.
  • IGFBP-1 in the normal human serum samples were quantitated using a nine-point calibrator curve with an analytical range from 0 to 500 ⁇ g/L, and quality controls (0.75, 3.50, 35.0 & 350 ⁇ g/L) diluted in Porcine Diluent.
  • CRP in the normal human serum samples were quantitated using an eight-point calibrator curve with an analytical range from 0 - 6 ⁇ g/mL. Bio-Rad controls were used as the assay quality controls. Results from the normal serum sample testing showed that the reference range for total adiponectin was from 3.23 - 25.77 ⁇ g/mL. Results from the normal serum sample testing showed that the reference range for total IGFBP-1 was from 0.03-284 ⁇ g/L with an average concentration of 41 .9 ⁇ g/L.
  • the serum pool prepared for testing the spike recovery of the IGFBP-1 assay had a concentration of 28.2 ⁇ g/L and the spiked serum sample tested at 34.4 ⁇ g/L. For IGFBP-1 , the measured spike concentration was 6.2 ⁇ g/L, which was 62% of the 10 ⁇ g/L spike concentration.
  • a BV-TAG Plus anti-Human Adiponectin monoclonal antibody was prepared and diluted to a working concentration of 10 ⁇ g/mL in anti-HAMA diluent.
  • the prebound monoclonal antibody beads were prepared at a concentration of 0.25 mg/mL.
  • the pre-bound beads were diluted to a working concentration of 0.25 mg/mL in anti-HAMA antibody diluent.
  • Prior to analysis all control and test samples were diluted to 1 :300 in DILUENT with 0.5% BSA.
  • adiponectin quality controls were prepared at trilevels of 0.18, 1 .8, and 18 ⁇ g/mL in DILUENT with 0.5% BSA.
  • the controls Prior to adding to the microplate, the controls were diluted to 1 :300 dilution in DILUENT with 0.5% BSA. Calibrators were prepared in DILUENT with 0.5% BSA to the following levels of 0.0, 0.41 , 1 .02, 2.56, 6.4, 16, 40, and 100 ng/mL.
  • An antibody master mix was prepared by adding equal volumes of biotinylated monoclonal antibody beads and BV-TAG Plus labeled monoclonal antibody into a microfuge tube (3.0 mL/each), prior to adding to the microplate.
  • test samples were diluted to 1 :300 in DILUENT with 0.5% BSA. Then 50 ⁇ of each calibrator, control, and test sample was added to the plate wells following the plate layout. 50 ⁇ of antibody master mix was then added to wells containing calibrator, control, and test samples per plate layout. The plate was sealed and shaken at room temperature for 60 ⁇ 5 minutes.
  • each assay plate was washed 2X with 150 ⁇ / ⁇ of WD-DILUENT solution.
  • the microplate was evaluated on one M1 MR Analyzer (Wellstat Diagnostics, LLC) using the plate protocol Adiponectin: 100 ⁇ assay volume and 50 ⁇ draw volume.
  • the assay plates were analyzed and data collected. The concentrations generated from the testing were added to the
  • biotinylated monoclonal antibody coated beads were prepared using Dynabeads ® Streptavidin (SA) beads that were pre-bound with biotinylated monoclonal antibody M5 at 8 ⁇ g of biotinylated monoclonal antibody per mg of beads.
  • the prebound beads were diluted to a working concentration of 0.4 mg/mL in Conjugate Buffer.
  • the BV-TAG Plus Conjugated Anti-CK18 monoclonal antibody clone M30 (0.73 mg/mL) was diluted to a working concentration of 5 ⁇ g/mL in Conjugate Diluent.
  • the K-18 standards and controls were diluted in Porcine Diluent.
  • the Standards, Samples and Controls were tested undiluted in duplicate at RT with the 60 ⁇ 5 minute incubation. Then 50 ⁇ of the Standards, Controls, and patient samples were added to the wells of the plate.
  • Equal volumes (3,500 ⁇ ) of capture and detector reagents were combined to form a master mix. Then 75 ⁇ of the master mix was added to a 96- well plate. The plate was then covered with a plate cover and incubated for 60 ⁇ 5 minutes at room temperature with shaking. Following the assay incubation, the plates were washed 2X with 150 ⁇ _ WD-DILUENT. The beads were resuspended with 100 ⁇ WD-DILUENT. The plates were evaluated on one M1 MR analyzer. The raw data was analyzed using SOFTmax Pro GxP with a 5 parameter logistic curve fit. The concentrations generated from the testing were added to the Combined Assay Results Tables 2A-2C.
  • the CK-18 Controls were prepared in Porcine Diluent at concentrations of 150, 750 and 1500 U/L from a stock of 41 ,800 Units/L*. (*Recombinant Antigen stock concentration adjusted to 41 ,800 U/L by applying offset of 2.09 to vendor- supplied concentration of 20,000 U/L). The controls were prepared fresh for each assay.
  • the stock BV-TAG Plus labeled anti-CK-18 monoclonal antibody clone M5 was diluted to 1 :279 with Conjugate Buffer to a working concentration of 2 ⁇ g/mL.
  • the stock biotinylated monoclonal antibody anti-CK-18 Clone M6 coated beads were diluted to a working concentration of 0.2 mg/mL.
  • Reagent Master Mixes were made by adding the same volume of diluted anti-CK18 monoclonal antibody Clone M5 to the tubes containing the diluted biotinylated monoclonal antibody anti-CK-18 Clone M6 coated beads as noted above. 25 ⁇ _ of each CK-18 M65 Standard, Control, Porcine Diluent for 0, and noted serum samples were added to their respective wells.
  • biotinylated anti-Human CRP monoclonal antibody C3 was pre- bound to Dynabeads ® M280 streptavidin (SA) beads at 10 ⁇ g per mg of beads. The pre-bound beads were then diluted to 0.3 mg/mL in anti-HAMA Antibody Diluent.
  • the BV-TAG Plus conjugated anti-Human CRP monoclonal antibody CRP135 was diluted to 10 ⁇ g/mL in anti-HAMA Antibody Diluent.
  • the master-mix was prepared just prior to addition to the assay plate by adding equal volumes of the biotinylated monoclonal antibody/beads (0.30 mg/mL) and BV-TAG Plus monoclonal antibody (10 ⁇ g/mL) at a final volume sufficient for addition of 50 ⁇ / ⁇ in each assay plate based on the plate layout.
  • the CRP calibrators were prepared at concentrations of 0.0, 0.0043, 0.013, 0.04, 0.123, 0.37, 1 .1 1 , and 6 ⁇ g/mL in anti-HAMA Antibody Diluent.
  • Bio-Rad controls were used as the assay quality controls.
  • the material was supplied as a liquid serum sample containing a target concentration of CRP.
  • each control was diluted 1 :50 in anti-HAMA Antibody Diluent.
  • Each test sample was diluted 1 :50 in anti-HAMA Antibody Diluent prior to addition to the assay plates.
  • Each assay plate had one set of eight calibrators and one set of three controls along with test samples in duplicate. 50 ⁇ - ⁇ /vell of each calibrator, diluted (1 :50) quality control and test sample was added to the microplate according to the plate layout. 50 ⁇ - ⁇ /vell of master mix was added to the microplate. The plate was sealed and shaken for 60 ⁇ 5 minutes at room temperature on the Micromix 5 shaker (DPC/Siemens) set at Form 8 Amp 6. Following incubation, each assay plate was washed 2X with 125 ⁇ - ⁇ /vell of WD-DILUENT. The washed bead complex was resuspended in 100 ⁇ - ⁇ /vell of WD-DILUENT.
  • Each of the four plates from the first test day were evaluated on different M 1 MR Analyzers using the following plate protocol including CRP: 100 ⁇ _ assay volume; 50 ⁇ _ draw volume.
  • Each of the two plates from the second test day were evaluated on two M1 MR Analyzers used on the initial test day with the exact same assay protocol as above.
  • the raw data was analyzed using SOFTmax Pro GxP with a 4 parameter logistic curve fit.
  • the concentrations generated from the testing were added to the Combined Assay Results Tables 2A-2C.
  • the concentrations generated from normal patient serum samples were added to Table 3.
  • IGFBP-1 Insulin-Like Growth Factor Binding Protein 1
  • the biotinylated anti-human IGFBP-1 monoclonal antibody was prebound to Dynabeads ® M280 streptavidin (SA) beads at a ratio of 10 ⁇ g biotinylated antibody per mg of beads.
  • the beads were diluted to a working concentration of 0.15 mg/mL in anti-HAMA Antibody Diluent.
  • the BV-TAG Plus conjugated anti-Human IGFBP-1 monoclonal antibody was diluted to a working concentration of 10 ⁇ g/mL in anti-HAMA Antibody Diluent.
  • Master mix was prepared just prior to addition to the assay plate by adding equal volumes of the biotinylated monoclonal antibody/beads (0.15 mg/mL) and BV-TAG Plus monoclonal antibody (10 ⁇ g/mL) at a final volume sufficient for addition of 50 ⁇ / ⁇ in each assay plate based on the plate layout.
  • the IGFBP-1 calibrators were prepared at concentrations of 0.0, 0.10, 0.25, 1 .00, 5.00, 25.0, 100, 250, and 500 ⁇ 9/ ⁇ _ in Porcine Diluent.
  • the IGFBP-1 antigen (Hytest Cat. # 8IGB1 ) was supplied as a lyophilized powder (0.1 mg/tube) stored at -20°C.
  • the IGFBP-1 powder was reconstituted with 1 ml. of Dl water to prepare a 100 ⁇ g/mL stock solution (Stock I). The stock was aliquoted and stored at -20°C.
  • the 100 ⁇ g/mL (Stock I) was diluted to 2000 ⁇ g/L (IGFBP-1 Intermediate) in Porcine Diluent.
  • the IGFBP-1 controls were prepared at levels of 0.75, 3.5, 35.0, and 350 ⁇ g/mL in Porcine Diluent.
  • the IGFBP- 1 controls were prepared from the IGFBP-1 Intermediate (2000 ⁇ 9/ ⁇ ). The volume prepared was sufficient for two assay plates.
  • test samples were prepared and then each test sample was added to the assay plate undiluted with 50 ⁇ _ per well in duplicate.
  • Each assay plate had one set of nine calibrators and one set of four controls along with test samples analyzed in duplicate.
  • the assay plates were evaluated on the M1 MR Analyzer.
  • the raw data was analyzed using SOFTmax Pro GxP with a 4 parameter logistic curve fit.
  • the concentrations generated from the testing were added to the Combined Assay Results Tables 2A-2C.
  • the concentrations generated from normal patient serum samples were added to Table 3.
  • the principle objective of the statistical analyses was to determine if biomarkers of cell injury and insulin metabolism provide unique predictive information about patient NASH status (absent, present) beyond the predictive information provided by common measures of liver disease progression and obesity.
  • three biomarkers of cell injury adiponectin, full-length keratin-18, and caspase-3-cleaved keratin-18
  • one biomarker of insulin metabolism insulinlike growth factor binding protein 1
  • NAS ⁇ 4, NAS ⁇ 4 alanine aminotransferase and body mass index as predictors of patient NASH status
  • ADPN adiponectin
  • IGFBP-1 insulin-like growth factor binding protein 1
  • ALT aminotransferase
  • BM I body mass index
  • Binomial generalize estimating equation (GEE) models (Hardin J.W., Hilbe, J.M. (2003), Generalized Estimating Equations, Chapman & Hall/CRC, New York, New York.) were used to conduct the second set of analyses.
  • the outcome variable for each GEE analysis was binary (i.e. 0 or 1 ).
  • the value 1 was assigned if the patient's biopsy NAS was less than 4 (NAS ⁇ 4), and the value 0 assigned if the patient's biopsy NAS was greater than or equal to 4 (NAS>4).
  • ADPN ADPN, caspase-3-cleaved K-18 (C3C K-18 or M30 antigen), and full-length K-18 (K-18 or M65 antigen), the biomarker related to insulin
  • the dependent variable in the regression analysis was a binary indicator of patient NASH status.
  • the value 1 was assigned if the patient's biopsy NAS was less than 4 (NAS ⁇ 4), and the value 0 assigned if the patient's biopsy NAS was
  • a receiver operator characteristic curve (ROC) (Huber, 1967) was generated based on the multivariate binomial GEE regression predicted probabilities for NAS ⁇ 4.
  • An optimal probability cut-point classification rule was then established to classify the liver biopsies according to NASH status (NAS ⁇ 4 versus NAS ⁇ 4).
  • the level of diagnostic agreement between the model-based liver biopsy NASH status classifications and the hepatologist's biopsy NASH status classifications was evaluated in terms of classification sensitivity and specificity, as well as in terms of the false positive and false negative error rates of the model based NASH status classifications.
  • ⁇ the p value is for the test that the geometric means are equal for those patients with NAS ⁇ 4 and those patients with NAS ⁇ 4.
  • the odds ratios from the univariate binomial GEE analyses are presented in Table 5.
  • the odds ratio compares the odds of a NAS ⁇ 4 for patients who have measurements at the 3 rd quartile of the predictor variable measurement distribution relative to the odds of a NAS ⁇ 4 for patients who have measurements at the 1 st quartile of predictor variable measurement distribution.
  • NASH status was significantly associated with all predictor variables other than ALT. With the exception of the association between ADPN and patient NASH status, all associations were in an inverse direction, meaning that patients who have large values for the predictor variable are less prone to have a NAS ⁇ 4.
  • the relationship between the value of the predictor variable and the predicted probability of a NAS ⁇ 4 is graphically summarized in FIG. 2 for each predictor variable.
  • Odds ratios compare the odds of NAS ⁇ 4 for a patient who has a value at the 3 rd quartile of the predictor variable measurement distribution relative to the odds of NAS ⁇ 4 for a patient who has a value at the 1 st quartile of the predictor variable measurement distribution.
  • the individual predictor variables are ranked according to the strength of the partial association between the predictor variable and patient NASH status (NAS ⁇ 4, NAS ⁇ 4).
  • the criterion for the ranking of predictors is based on the magnitude of difference between the observed Wald chi-squared statistic (see Table 7) and the expected Wald chi-squared statistic under the null hypothesis of no partial association.
  • BM I and ADPN are the predictor variables that rank the highest in relative importance, followed by C3C K-18, IGFBP- 1 , and K-18.
  • the model C-statistic is 0.95.
  • the C-statistic is a measure of the concordance between the model predictions and actual risk.
  • the value of the C-statistic ranges from 0.5 to 1 .
  • a C-statistic equal to 0.5 indicates that the model classifies the observation units into their true groups no better than a classification scheme that is based on the flip of a fair coin, while a C-statistic equal to 1 indicates the model can classify the observation units into their true groups perfectly.
  • NAS ⁇ 4 was 0.95.
  • the optimal cut-point probability was 0.174 for correctly
  • variable ALT Since the variable ALT was of only marginal associated with patient
  • the model C-statistic is 0.95. This value is equal to the value of the C-statistic of the model that included the set of predictors, plus ALT.
  • Table 9 Regression coefficient estimates and associated standard error for the multivariate binomial GEE regression model specified in equation 2 of the statistical methods
  • the area under the receiver operator characteristic curve (ROC) that was generated based on the multivariate GE regression predictions for the probability of having a NAS ⁇ 4 was 0.95.
  • the optimal cut-point probability was 0.174 for correctly classifying the patients according to their clinically determined patient NASH status (NAS ⁇ 4 versus NAS ⁇ 4).
  • the sensitivity of the model-based patient NASH status classification rule was 1 .0. Out of the 13 biopsies that had a NAS ⁇ 4, all were correctly classified.
  • the specificity of the model- based patient NASH status classification rule was 0.87. Out of the 53 liver biopsies in which the hepatologist gave the biopsy a NAS ⁇ 4, 7 biopsies were misclassified.
  • the false positive error rate of the model-based patient NASH status classification rule was 0.13, while the false positive error rate was 0.
  • a nomogram for predicting the probability that a patient has a NAS ⁇ 4 is presented in Table 10.
  • the nomogram was developed based on the model parameter estimates of the model that is specified in equation 2.
  • the nomogram in Table 10 is based on a points system. To use the nomogram you simply add up the points associated with each predictor variable (ADPN, C3C K-18, K-18, IGFBP-1 , and BMI) to determine the patient's total number of points. In the lower right portion of the nomogram, two columns of numbers are underlined. In the left column of the underlined area, the total number of points are listed, and in right column of the underlined area, the corresponding predicted probabilities for NAS ⁇ 4 are listed. It should be noted that since no external validation of the model in equation 2 was performed, at this time the utility of the nomogram is specific to this particular set of patients.
  • Table 10 Nomogram for predicting a patient's probability of having a NAS ⁇ 4 based on the patient's ADPN, C3C K-18, K-18, 1 GFBP-1 and BMI values.
  • Tables 2A-2C An alternative framework for evaluating the NASH clinical trial data shown in the Combined Assay Results Tables 2A-2C is to correlate the change in NAS with changes in serum analytes, regardless of the starting or ending NAS value.
  • Table 1 1 below divides patients into three groups according to the change in NAS: (1 ) Reduction in NAS ⁇ 1 , (2) Reduction in NAS ⁇ 1 , and (3) Reduction in NAS ⁇ 2, with the absolute and percent change (from baseline) of each measured serum biomarker.
  • Table 1 1 indicates that downward percent changes in caspase-3-cleaved K-18, full-length K-18, IGFBP-1 , CRP and ALT increase as a function of the downward change in NAS value.
  • Tables 12-17 The data generated by the assays completed for each of the different biomarkers was broken down further in Tables 12-17 based on the same patient serum samples from Example 1 .
  • each of Tables 12-17 provides results following the change in NAS values for each of the biomarkers assayed on the panel: Adiponectin (Table 12); caspase-3-cleaved keratin-18 (Table 13); full-length keratin-18 (Table 14); IGFBP-1 (Table 15); C-Reactive Protein (Table 16); and ALT (Table 17).
  • NAS values have been left in the tables to corroborate the collected data from Tables 2A-2C and Table 1 1 .
  • the data generated from these analyses demonstrate that the assay panel of the disclosure is useful not only for assigning a likely NAS value at a single time point, but can be used to detect changes in liver histology over time, regardless of whether the final result is an NAS value ⁇ 4.
  • a patient with an initial NAS of 6.5 and a later (e.g., 1 year) value of 4.5 can be identified by the panel of the disclosure to provide guidance for continued treatment, as the trajectory of liver disease is improving even though the patient still has a clinical diagnosis of NASH (NAS>4).
  • liver histology correlating with changes in NAS numerical values
  • Use of the diagnostic panel to detect changes in liver histology provides an important tool for conducting efficient clinical studies to identify effective therapies, or disprove ineffective therapies, and for providing information to encourage patients to persist in lifestyle changes with beneficial effects on liver status.

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Abstract

La probabilité qu'un sujet ait une stéatohépatite non alcoolique histologique (NASH) significativement active ou un changement d'une NASH histologique peut être prédit(e) en mesurant le niveau d'au moins quatre biomarqueurs sélectionnés dans le groupe composé de l'adiponectine, la kératine 18 pleine longueur, la kératine clivée par la caspase-3, la protéine C réactive, la protéine 1 de liaison du facteur de croissance analogue à l'insuline, et l'alanine amino transférase dans un échantillon dérivé du sujet, et en analysant le niveau desdits quatre biomarqueurs par rapport à des valeurs de référence pour ces biomarqueurs. Cette approche peut être utilisée pour le dépistage de la présence ou de l'absence de NASH, pour la surveillance de la progression ou de la régression de la NASH au cours du temps, ou pour surveiller l'efficacité d'une thérapie de traitement de la NASH.
PCT/US2012/057826 2011-09-28 2012-09-28 Panel de dosage pour la stéatohépatite non alcoolique Ceased WO2013049509A1 (fr)

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WO2014049131A1 (fr) * 2012-09-28 2014-04-03 Université d'Angers Test sanguin précis pour le diagnostic non invasif de la stéatohépatite non alcoolique
CN112129950A (zh) * 2020-09-11 2020-12-25 武汉生之源生物科技股份有限公司 一种检测白介素6的磁微粒化学发光试剂盒
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014049131A1 (fr) * 2012-09-28 2014-04-03 Université d'Angers Test sanguin précis pour le diagnostic non invasif de la stéatohépatite non alcoolique
EP3586141B1 (fr) * 2017-02-27 2023-01-18 Université d'Angers Diagnostic non-invasif de la stéatohépatite non alcoolique (nash) fibrosante
CN112129950A (zh) * 2020-09-11 2020-12-25 武汉生之源生物科技股份有限公司 一种检测白介素6的磁微粒化学发光试剂盒
WO2022093757A1 (fr) * 2020-10-30 2022-05-05 The General Hospital Corporation Kits, réactifs et procédés d'évaluation de maladies hépatiques
CN116959734A (zh) * 2023-05-17 2023-10-27 南方医科大学南方医院 一种代谢相关脂肪性肝病发病的预测方法及系统
CN117347626A (zh) * 2023-12-06 2024-01-05 安徽惠邦生物工程有限公司 人胰岛素样生长因子结合蛋白-1化学发光免疫分析试剂盒及其制备方法
CN117347626B (zh) * 2023-12-06 2024-03-12 安徽惠邦生物工程有限公司 人胰岛素样生长因子结合蛋白-1化学发光免疫分析试剂盒及其制备方法

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