Use of adiponectin, ferritin, MMP9, and transferrin for monitoring liver disease treatment with ranifibranol

Abstract

This disclosure relates to a method for evaluating the efficacy of treatment with ranifibranol in patients with liver disease, the method comprising: a) measuring in vitro the levels of a combination of biomarkers consisting of adiponectin, ferritin, MMP9 and transferrin in a biological sample obtained from a patient; and b) evaluating the efficacy of treatment with ranifibranol according to the levels measured in step a).

Description

【Technical Field】 【0001】 The present disclosure relates to a method for evaluating the efficacy of treatment with the investigational drug lanifibranor in patients with liver disease. The present disclosure also relates to a method for treating liver disease, which includes the step of evaluating the efficacy of lanifibranor treatment. 【Background Art】 【0002】 Non-alcoholic steatohepatitis (NASH) is a major cause of chronic liver disease. Its diagnosis and characterization currently rely on histological examination, and liver biopsy is still required for the diagnosis and evaluation of treatment response. However, considering the invasiveness of liver biopsy, as well as the drawbacks of histological evaluation such as high sampling variability, poor inter-observer assessment, and the possibility of complications, there is an urgent need for alternative diagnostic methods. Identifying biomarker signatures for non-invasively evaluating histological response would be an important step in overcoming the drawbacks of currently used diagnostic methods and promoting NASH treatment. 【0003】 Lanifibranor, a pan-peroxisome proliferator-activated receptor (PPAR) agonist, is a promising investigational compound that regulates major metabolic, inflammatory, and fibrogenic pathways and showed a therapeutic effect on both NASH resolution and fibrosis improvement in the Phase 2b NATIVE trial. In this trial, promising results were shown that the proportion of patients with active NASH whose steatosis-activity-fibrosis (SAF)-A score decreased by at least 2 points without deterioration of fibrosis was significantly higher after treatment with 1200 mg of lanifibranor per day compared to placebo treatment. The secondary endpoints of this trial included NASH resolution and fibrosis improvement, NASH resolution without fibrosis deterioration, and fibrosis improvement without NASH deterioration, according to the NASH Clinical Research Network (NASH-CRN). The results of this trial were more favorable in the lanifibranor group than in the placebo group. 【Summary of the Invention】 [Problems that the invention aims to solve] 【0004】 In this context, the inventors' objective was to identify the biological signature of histological responders among NASH patients treated with ranifibranol. Identifying and developing biomarker signatures can, in fact, facilitate the assessment of response to treatment and help identify patients most likely to receive clinical benefit from treatment. [Means for solving the problem] 【0005】 This disclosure relates to a method for evaluating the effectiveness of treatment with ranifibranol in patients with liver disease, and the method is as follows: a) To measure in vitro the levels of a combination of biomarkers consisting of adiponectin, ferritin, MMP9, and transferrin in biological samples obtained from the above patients; b) Evaluate the effectiveness of treatment with ranifibranol according to the level measured in step a) and Includes. 【0006】 In some embodiments, adiponectin and ferritin levels are measured before the initiation of treatment with ranifibranol. 【0007】 In some embodiments, MMP9 and transferrin levels are measured before the initiation of treatment with ranifibranol and after at least three months of treatment with ranifibranol. 【0008】 In some embodiments, the levels measured in step a) are used to obtain a score to which the patient belongs to a determined or uncertain prognosis class. In some embodiments, the determined prognosis class includes a class of patients predicted to respond to ranifibranol treatment and a class of patients predicted to be non-responders to ranifibranol treatment. In some embodiments, the score is obtained by combining the levels measured in step a) in a mathematical function. In some embodiments, the mathematical function is a binary logistic regression. In some embodiments, the method includes comparing the score with a first calculated cutoff value, below which non-response to ranifibranol treatment is predicted. In some embodiments, the method includes comparing the score with a second calculated cutoff value, above which response to ranifibranol treatment is predicted. 【0009】 In some embodiments, the biological sample is a sample of biological fluid. In some embodiments, the biological fluid is blood, serum, or plasma. 【0010】 In some embodiments, the liver disease is non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, compensated or decompensated cirrhosis, hepatic fibrosis, fatty liver disease, acute liver failure, or acute exacerbation of chronic liver failure. 【0011】 This disclosure also relates to a system for evaluating the effectiveness of treatment with ranifibranol in patients with liver disease, and the system is a) Means for measuring the levels of a combination of biomarkers consisting of adiponectin, ferritin, MMP9, and transferrin in a biological sample obtained from the above patient, or for receiving such measurement data; b) A data processing means configured to evaluate the effectiveness of treatment with ranifibranol in the patient according to the measured levels of the above combination of biomarkers. Includes. 【0012】 This disclosure also relates to a combination of biomarkers consisting of adiponectin, ferritin, MMP9, and transferrin for use in a method for evaluating the efficacy of treatment with ranifibranol in patients with liver disease. 【0013】 This disclosure also relates to a method for treating liver disease in the subject, and the method is a) Before initiating treatment, measure in vitro the levels of a combination of biomarkers consisting of adiponectin, ferritin, MMP9, and transferrin in biological samples obtained from the above patients; b) The subjects described above shall be administered an effective dose of ranifibranol daily for at least three months; c) After 3 months of treatment, measure the levels of MMP9 and transferrin in the above-mentioned biological samples in vitro; d) Evaluate the effectiveness of treatment with ranifibranol according to the levels measured in steps a) and c); e) If the evaluation performed in step d) predicts the response to ranifibranol treatment, or if the prognosis cannot be determined, the subject shall continue to receive an effective dose of ranifibranol for at least another 3 months. Includes. [Brief explanation of the drawing] 【0014】 [Figure 1] Sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) are indicated according to the E1 score. [Modes for carrying out the invention] 【0015】 This disclosure relates to a method for evaluating the effectiveness of treatment with ranifibranol in patients with liver disease, and the method is as follows: a) Measuring in vitro the levels of a combination of biomarkers consisting of adiponectin, ferritin, MMP9, and transferrin in a biological sample obtained from a patient; b) Evaluating the effectiveness of treatment with ranifibranor according to the levels measured in step a); and including. 【0016】 In the context of the present disclosure, MMP9 represents matrix metallopeptidase 9. 【0017】 In the context of the present disclosure, the terms "combination of biomarkers", "biomarker signature", and "biomarkers signature" are used interchangeably. 【0018】 As used herein, "evaluation of the effectiveness / effect of treatment" refers to the determination of the clinical status of a subject undergoing treatment. The treatment may be preventive, such as in cases where there is a predisposition to a disease, or therapeutic, such as in cases of a diagnosed disease. The effectiveness of treatment may be evaluated, for example, by determining the status of the patient at various time intervals. The status of the patient is particularly evaluated before the first treatment, and after this first treatment (e.g., each time a new treatment is received), at regular (or irregular) time intervals. And by comparing the status of the patient evaluated at these various intervals, potential changes can be identified. The status of the patient may be evaluated based on observations and / or measurements made using various tools. 【0019】 As used herein, "treatment" refers to the process, action, application, therapy, etc., by which a patient receives assistance, particularly medical or veterinary assistance, for the purpose of directly or indirectly improving the patient's condition. 【0020】 As used herein, “patient” or “subject” refers to a human individual or a non-human animal. A patient is, for example, a human or animal that is susceptible to or has a liver disease. A patient is preferably human. A patient may be a child (a human patient under 18 years of age) or an adult (a human patient over 18 years of age). In the context of this disclosure, the terms “patient” and “subject” are interchangeable. 【0021】 As used herein, “measurement” is understood to mean the quantitative characterization of a physical object or entity or a number (group or group) thereof or their functions, or the quantitative characterization of a physical or chemical process, which involves assigning a quantity, value, e.g., numerical value or number, specific to the object or entity or number or function or process by comparison with units and with other objects or entities or numbers or functions or processes. The measurement is preferably consistent with methods known in the art or with international metrological guidelines. 【0022】 As used herein, “quantification” or “quantification” is understood to mean assigning a numerical or numerical and unital physical quantity to a physical object or entity, or a number (group or group) of such objects or entities, or their functions, or to a physical or chemical process, in comparison to other objects or entities. Advantageously, “quantification” or “quantification” is a measurement or an essential part of measurement. Measurements are subject to uncertainty, which may represent random and systematic errors in the measurement procedure. Those skilled in the art are aware of this and can handle these errors in consideration of the measurement or quantification being applied. 【0023】 As used herein, ranifibranol is a pan-PPAR agonist of formula {4-[1-(1,3-benzothiazole-6-ylsulfonyl)-5-chloroindole-2-yl]butanoic acid; CAS 927961-18-0}. Advantageously, the term “ranifibranol” also includes the deuterated form of ranifibranol or its pharmaceutically acceptable salts. The deuterated form of ranifibranol may be one disclosed in International Application WO2020 / 021215 (the disclosure of which is incorporated by reference). 【0024】 In some embodiments, the biomarker whose level is measured in step a) is a protein biomarker. Advantageously, the biomarker is a serum biomarker. 【0025】 In some embodiments, adiponectin and ferritin levels are measured before the initiation of treatment with ranifibranol. 【0026】 In some embodiments, MMP9 and transferrin levels are measured before the initiation of treatment with ranifibranol and after at least three months of treatment with ranifibranol. Advantageously, MMP9 and transferrin levels are measured after at least four months, advantageously at least five months, advantageously at least six months, advantageously at least seven months, advantageously at least eight months, advantageously at least nine months, advantageously at least ten months, advantageously at least eleven months, advantageously at least twelve months, or longer of treatment with ranifibranol. 【0027】 In some embodiments, the levels measured in step a) are used to obtain a score to which the patient belongs to a confirmed or uncertain prognosis class. In some embodiments, the confirmed prognosis class includes a class of patients predicted to respond to ranifibranol treatment and a class of patients predicted to not respond to ranifibranol treatment. 【0028】 In some embodiments, the score is obtained by combining the levels measured in step a) in a mathematical function. In some embodiments, the mathematical function is a binary logistic regression. 【0029】 In some embodiments, the method includes comparing the score with a first calculated cutoff value, below which non-response to ranifibranol treatment is predicted. In advantageous embodiments, the first calculated cutoff value is approximately 0.299. 【0030】 In some embodiments, the method includes comparing the score with a second calculated cutoff value, above which a response to ranifibranol treatment is predicted. More specifically, if the obtained score (hereinafter also referred to as the "E1 score") exceeds the second calculated cutoff value, it is predicted that NASH will disappear and liver fibrosis will improve by one stage or more. In advantageous embodiments, the second calculated cutoff value is approximately 0.592. 【0031】 In some embodiments, the biological sample is a sample of biological fluid. In some embodiments, the biological fluid is blood, serum, or plasma. Advantageously, the biological fluid is blood. 【0032】 In some embodiments, the liver disease is non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cirrhosis such as compensated or decompensated cirrhosis, liver fibrosis, fatty liver disease, acute liver failure, or acute exacerbation of chronic liver failure. 【0033】 This disclosure also relates to a system for evaluating the effectiveness of treatment with ranifibranol in patients with liver disease, the system being: a) Means for measuring the levels of a combination of biomarkers consisting of adiponectin, ferritin, MMP9, and transferrin in a biological sample obtained from a patient, or for receiving such measurement data; b) Data processing means configured to evaluate the effectiveness of treatment with ranifibranol in patients according to the measured levels of a combination of biomarkers. Includes. 【0034】 This disclosure also relates to a combination of biomarkers consisting of adiponectin, ferritin, MMP9, and transferrin for use in a method for evaluating the efficacy of treatment with ranifibranol in patients with liver disease. 【0035】 In some embodiments, the liver disease is non-alcoholic fatty liver disease, non-alcoholic steatohepatitis (NASH), cirrhosis such as compensated or decompensated cirrhosis, liver fibrosis, fatty liver disease, acute liver failure, or acute exacerbation of chronic liver failure. 【0036】 In some embodiments, treatment with ranifibranol eliminates NASH and improves liver fibrosis by one or more stages. 【0037】 This disclosure also relates to a method for treating liver disease in the subject, the method being: a) In vitro measurement of the levels of a combination of biomarkers consisting of adiponectin, ferritin, MMP9, and transferrin in a biological sample obtained from the patient before the start of treatment; b) The subjects shall be administered an effective dose of ranifibranol daily for at least three months; c) After 3 months of treatment, measure the levels of MMP9 and transferrin in the target biological sample in vitro; d) Evaluate the effectiveness of treatment with ranifibranol according to the levels measured in steps a) and c), thereby predicting a response or non-response to ranifibranol treatment; e) If the assessment performed in step d) predicts a response to ranifibranol treatment, or if a diagnosis cannot be made, the subject should continue to receive an effective dose of ranifibranol for at least another 3 months. Includes. 【0038】 In embodiments involving the administration of a therapeutically effective dose of ranifibranol to a patient in need of treatment, “therapeutically effective,” “effective dose,” “therapeutably effective dose,” or “pharmaceutically effective” means the amount of ranifibranol needed to inhibit or reverse a disease state (for example, to treat liver disease). The determination of the effective dose specifically depends on factors such as the safety and efficacy of the drug. These factors will vary depending on other factors such as potency, relative bioavailability, patient weight, severity of adverse side effects, and preferred method of administration. Safety may be determined using methods well known to those skilled in the art. Efficacy may be determined using the same guidelines. Therefore, a pharmaceutically effective dose is the amount that a clinician deems safe and effective. 【0039】 The dose may be adjusted as appropriate to achieve the desired level locally or systemically, depending on the method of administration. If the patient's response is insufficient with such a dose, a higher dose (or an effective higher dose via a different, more localized delivery route) may be used, within the limits of the patient's tolerance. Multiple doses may be administered daily to achieve an appropriate systemic level of ranifibranol. An appropriate systemic level can be determined, for example, by measuring the patient's peak or sustained plasma level of the drug. The terms "dosage" and "dose" are used interchangeably herein. 【0040】 In some embodiments, ranifibranol is administered in a daily dose of approximately 400 mg to approximately 1200 mg, according to step b) of the method for treating liver disease in a subject. Advantageously, ranifibranol is administered in a daily dose of approximately 400 mg, advantageously, approximately 500 mg, advantageously, approximately 600 mg, advantageously, approximately 700 mg, advantageously, approximately 800 mg, advantageously, approximately 900 mg, advantageously, approximately 1000 mg, advantageously, approximately 1100 mg, and advantageously, approximately 1200 mg. In some embodiments, ranifibranol is administered to the patient with food. In some embodiments, ranifibranol is administered to the patient under fasting conditions. 【0041】 In some embodiments, ranifibranol is used in vivo. Depending on the intended in vivo administration method, ranifibranol may be administered in solid, semi-solid, or liquid dosage forms. In some embodiments, ranifibranol is administered in solid dosage form. Examples of solid dosage forms include tablets, capsules, stick packs, sachets, lozenges, powders, pills, or granules. Preferred solid dosage forms include tablets, capsules, and stick packs, with tablets being particularly preferred. Advantageously, ranifibranol is administered in unit dosage forms suitable for a single dose of a precise amount. Depending on the desired formulation, ranifibranol can be formulated into a pharmaceutical composition comprising ranifibranol and one or more pharmaceutically acceptable excipients. The selection of excipients will largely depend on factors such as the specific administration method, the effect of the excipients on solubility and stability, and the properties of the dosage form. The pharmaceutical compositions of the present invention can be prepared by conventional methods described in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995) (incorporated herein by reference), etc. In some embodiments, the pharmaceutically acceptable excipients include two or more of the following: binders, disintegrants, fillers, flow promoters, lubricants, and surfactants. In some embodiments, the pharmaceutically acceptable excipients include binders, disintegrants, fillers, flow promoters, lubricants, and surfactants. 【0042】 In some embodiments, the pharmaceutical composition contains 200 mg to 1200 mg of ranifibranol. Exemplary pharmaceutical compositions contain 200 mg, 400 mg, 600 mg, 800 mg, 1000 mg, or 1200 mg of ranifibranol. 【0043】 In some embodiments, ranifibranol is administered in a daily dose of approximately 400 mg to approximately 1200 mg. 【0044】 In any of the embodiments described above, ranifibranol may be in crystalline form, either on its own or in the pharmaceutical composition. 【0045】 Administration during in vivo treatment may be by any route, including oral, parenteral, intramuscular, intranasal, sublingual, intratracheal, inhalation, intraocular, vaginal, and rectal. Those skilled in the art will recognize that the route of administration varies depending on the disease being treated. Advantageously, ranifibranol or a pharmaceutical composition containing ranifibranol may be administered to the patient orally, parenterally, or topically. In one embodiment, the pharmaceutical composition containing ranifibranol is administered orally. 【0046】 In some embodiments, according to step b) of the method described above, ranifibranol is administered to the subject daily in an effective dose for at least 3 months, preferably at least 4 months, preferably at least 5 months, preferably at least 6 months, preferably at least 7 months, preferably at least 8 months, preferably at least 9 months, preferably at least 10 months, preferably at least 11 months, preferably at least 12 months, or longer. 【0047】 In some embodiments, the levels of MMP9 and transferrin are measured in the biological sample of the subject after treatment for at least 3 months, preferably at least 4 months, preferably at least 5 months, preferably at least 6 months, preferably at least 7 months, preferably at least 8 months, preferably at least 9 months, preferably at least 10 months, preferably at least 11 months, preferably at least 12 months, or longer, according to step c) of the method described above. 【0048】 In some embodiments, the evaluation in step d) is performed by obtaining a score (also called the E1 score) from the levels measured in steps a) and c), and comparing the score with (i) a first calculated cutoff value, below which a non-response to ranifibranol treatment is predicted, and (ii) a second calculated cutoff value, above which a response to ranifibranol treatment is predicted. If the obtained score is below the first calculated cutoff value, a non-response (i.e., negative response) to ranifibranol treatment is predicted. If the obtained score is above the second calculated cutoff value, a response (i.e., positive response) to ranifibranol treatment is predicted. In particular, if the obtained score is above the second calculated cutoff value, it is predicted that NASH will disappear and liver fibrosis will improve by one or more stages. If the obtained score falls between the first calculated cutoff value and the second calculated cutoff value, the prognosis regarding the effectiveness of ranifibranol treatment cannot be determined. 【0049】 In a favorable embodiment, the first calculated cutoff value is approximately 0.299. In a favorable embodiment, the second calculated cutoff value is approximately 0.592. 【0050】 In some embodiments, according to step e) of the method described above, ranifibranol is administered to the subject in an effective dose for at least a further 3 months, preferably at least a further 4 months, preferably at least a further 5 months, preferably at least a further 6 months, preferably at least a further 7 months, preferably at least a further 8 months, preferably at least a further 9 months, preferably at least a further 10 months, preferably at least a further 11 months, preferably at least a further 12 months, or longer. 【0051】 In some embodiments, the duration of treatment with ranifibranol described in step e) depends on the duration of administration of ranifibranol in step b). In some embodiments, the measurement in step c) is performed after treatment with ranifibranol. If the treatment with ranifibranol in step b) is 3 months, the measurement in step c) is performed after 3 months of treatment. 【0052】 In some embodiments, the biological sample is a sample of biological fluid. In some embodiments, the biological fluid is blood, serum, or plasma. Advantageously, the biological fluid is blood. 【0053】 In some embodiments, the liver disease is non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, compensated or decompensated cirrhosis, hepatic fibrosis, fatty liver disease, acute liver failure, or acute exacerbation of chronic liver failure. 【0054】 In some embodiments, the disclosure also includes kits for evaluating the efficacy of treatment with ranifibranol, and kits for measuring levels of adiponectin, ferritin, MMP9, and transferrin obtained from biological samples, etc. As used herein, “kit” is intended to mean a package, assembly, or container of materials intended to assist in the use of the assays of the present invention. 【0055】 The kits of this disclosure typically comprise one or more containers containing one or more reagents useful for carrying out the invention. Examples of reagents useful for carrying out the invention include, but are not limited to, buffers, buffer salts, metal ions, chromogenic compounds, antibodies, enzymes, and fluorescent compounds. The kits of this disclosure may comprise one or more containers containing adiponectin, ferritin, MMP9, and transferrin, or other compounds that can be used as reference standards. The kits of this disclosure may comprise a container containing one or more antibodies, the antibodies bound to a detectable moiety. The detectable moiety may be any known to those skilled in the art, and may be, for example, enzymes (peroxidase, luciferase, etc.), other proteins (green fluorescent protein, etc.), optically detectable compounds (fluorophores, chromophores, etc.), members of a binding pair (biotin / streptavidin, etc.), or other detectable moieties known to those skilled in the art. 【0056】 This disclosure is illustrated by the following embodiments. [Examples] 【0057】 Example 1: Materials and Method patient NATIVE was a multicenter, randomized, placebo-controlled phase 2b trial that investigated the safety and efficacy of ranifibranol in adult patients diagnosed with highly active non-cirrhotic NASH. Patients were eligible for enrollment if they were 18 years of age or older and had non-cirrhotic NASH (diagnosis required a steatosis-activity-fibrosis [SAF] grade of 1 or higher on liver biopsy for steatosis, hepatocyte ballooning degeneration, and intralobular inflammation). An SAF-A score of 3 or higher (the activity portion of the SAF scoring system incorporating scores for hepatocyte ballooning degeneration and intralobular inflammation) was also an eligibility criterion. Patients with fibrosis stage F4, classified according to both SAF and NASH Clinical Research Network (NASH CRN) criteria, were excluded from the trial. 【0058】 Patients were randomly assigned in a 1:1:1 ratio to receive either 800 mg or 1200 mg of ranifibranol or placebo once daily for 24 weeks. Liver biopsies were taken at baseline and at the end of treatment. The primary endpoint of the NATIVE trial was defined as a reduction of at least 2 points in the SAF-A score from baseline to week 24 (end of treatment) and no worsening of fibrosis. Secondary histological endpoints included: resolution of NASH (defined as balloon degeneration grade 0 and intralobular inflammation grade ≤ 1) and no worsening of fibrosis; improvement of at least one stage of fibrosis and no worsening of NASH (i.e., no worsening of steatosis, balloon degeneration, or intralobular inflammation); improvement of the Non-Alcoholic Fatty Liver Disease Activity Score (NAS) (defined as a decrease of at least 2 points from baseline to week 24) and no worsening of fibrosis; resolution of NASH and improvement of fibrosis stage by at least one (as a composite endpoint); changes in the scores of components of the SAF and NASH CRN scoring systems (steatosis, activity, inflammation, balloon degeneration, and fibrosis); and changes in the modified Ishak score. Non-histological secondary endpoints included changes in the serum biomarker panel for metabolism, inflammatory tissue damage, and fibrosis. 【0059】 Patients who received 800 mg or 1200 mg of ranifibranol per day were pooled, and those who underwent liver biopsy before or after treatment were selected (n=142). Post-treatment biopsies were considered if performed within 14 days of the last dose of ranifibranol. 【0060】 biomarkers A total of 71 notable biomarkers, including 65 laboratory parameters and six previously published diagnostic scores (FIB4, NAFLD fibrosis score (NFS), FIBC3, ABC3D, ELF, and MACK3), were measured at baseline and at end of treatment (EOT), i.e., week 24. The 65 laboratory biomarkers, as shown in Table 1, were primarily related to liver enzymes, lipid and glucose metabolism, inflammation, and liver fibrosis. 【0061】 [Table 1-1] [Table 1-2] 【0062】 First, we evaluated the accuracy of six diagnostic scores in predicting treatment response. Next, we incorporated baseline values ​​and changes under treatment for 65 laboratory parameters to derive new combined biomarker signatures. 【0063】 endpoint The following histological endpoints were considered according to the NASH-CRN criteria. Dissolution of NASH and improvement of fibrosis ≥ Stage 1 (E1) 【0064】 statistical methods 1 / Evaluation of available diagnostic scores We evaluated the ability of six published diagnostic scores (FIB4, NFS, FIBC3, ABC3D, ELF, and MACK3) available in the dataset to assess treatment response. For each score, we derived a multivariate model that included the baseline value of the diagnostic score, the absolute change between the baseline and EOT, and the relative change between the baseline and EOT. The discriminative ability of the models was assessed by the area under the receiver operating characteristic curve (AUROC). AUROC ranges from 0 to 1 and is interpreted as follows: 0.90 to 1.00 = very good, 0.80 to 0.90 = good, 0.70 to 0.80 = average, <0.70 = poor discriminative ability. Trials showing AUROC ≥ 0.8 are considered clinically noteworthy. 【0065】 2 / Development of a new signature for evaluating treatment response Overall, biomarker selection was performed using classical univariate analysis, principal component analysis (PCA), and sparse partial least squares discriminant analysis (sPLS-DA), and finally combined using logistic regression. 65 biomarkers were considered in three different ways: baseline value, absolute change between baseline and EOT, and relative change, resulting in 195 variables being considered in the analysis. 【0066】 First shortlist To identify biomarkers significantly associated with the evaluated endpoints, univariate analyses were performed using Student's t-test, Welch's t-test, or Wilcoxon-Mann-Whitney U tests as appropriate. Biomarkers with p-values ​​less than 10% were retained in the first shortlist. Furthermore, three PCA and sPLS-DA analyses were performed (one including all baseline biomarkers, one including all absolute changes between baseline and EOT, and the last including all relative changes between baseline and EOT). Box-Cox and Yeo-Johnson transformations were applied to normalize biomarkers in the PCA and sPLS-DA analyses. PCA was used to assess the association between data variability and response status. Where an association was found, the biomarker best represented on the PCA graph (those with a cosine squared greater than 0.35) was selected. sPLS-DA aimed to identify differentiater biomarkers between responders and non-responders in a supervised manner, and the top 15 were retained from this analysis. Ultimately, the biomarkers selected through seven analyses (univariate analysis, three PCAs, and three sPLS-DAs) constituted a first candidate shortlist for distinguishing between responders and non-responders. 【0067】 Second shortlist In this second stage, all candidate biomarkers from the first shortlist were introduced into the "final PCA" and "final sPLS-DA," ultimately yielding a second shortlist consisting of up to 15 biomarkers. Correlations between biomarkers in this second shortlist were controlled, and only biomarkers with a variance expansion coefficient of less than 4 were retained in the final biomarker shortlist. 【0068】 Logistic regression model The trial signature was constructed using logistic regression including biomarkers from a second shortlist. Model selection was performed using a stepwise method with the Akaike information criterion while controlling for interactions. Finally, the regression equation was derived from the logistic regression to calculate the probability of being a responder. 【0069】 3 / Signature accuracy evaluation Performance evaluation The discriminative ability of the signatures obtained for the E1 endpoint was evaluated using AUROC. Calibration (statistical agreement between predicted probabilities and observed values, i.e., predicted probabilities being close to 1 on average for responders and close to 0 for non-responders) was evaluated using the Brier score (BS). The BS ranges from 0 to 1, with lower BS indicating better calibration; i.e., a BS of 0 means perfect calibration. 【0070】 Use in clinical practice In clinical practice, signatures that predict histological response based on non-invasive testing may be useful in evaluating the efficacy of ranifibranol treatment. The 80% negative / positive predictive value (NPV / PPV) threshold can be used to exclude / confirm treatment response, yielding two cutoff values ​​indicating potential non-responders (i.e., below the lowest cutoff value), responders (i.e., above the highest cutoff value), or those in the grey zone (i.e., between the two cutoff values). A practical decision criterion based on this approach is shown in Figure 1. The reason for choosing to calculate the NPV / PPV threshold is that the evaluation of treatment response is at the individual level, not at the population level (where sensitivity and specificity are more relevant). 【0071】 The diagnostic performance of the calculated thresholds was evaluated based on sensitivity, specificity, negative and positive predictive values, size of the gray zone (smaller is better), and effectiveness of non-invasive diagnosis (the percentage of patients correctly classified among those outside the gray zone; higher is better). 【0072】 software I used the R software, which included packages such as FactoMiner, MixOmics, CAR, stats, pROC, DescTools, and ModelGood. 【0073】 Example 2: Results Test group In the native trial, from February 2017 to July 2019, 247 patients were randomly assigned to receive either ranifibranol 1200 mg (N=83), ranifibranol 800 mg (N=83), or placebo (N=81) orally once daily for 6 months. A total of 228 patients completed the trial, 77 in each ranifibranol group and 74 in the placebo group. Reasons for discontinuation of the trial regimen have been reported elsewhere. Ultimately, of the 154 patients treated with both doses of ranifibranol, 142 (patients treated with 800 mg or 1200 mg per day: N=70 and N=72, respectively) presented pre- and post-treatment liver biopsies within the time required for this study and were included in the analysis population for signature development. 【0074】 Demographic and clinical baseline characteristics of the entire population of 247 randomized treated patients have been reported elsewhere. Table 2 summarizes the baseline characteristics of the 142 patients retained in the analysis population for signature development. 【0075】 [Table 2] *The ± symbol indicates the mean ± standard deviation. To convert high-density lipoprotein (HDL) cholesterol values ​​to milligrams per deciliter, divide by 0.02586. To convert triglyceride values ​​to milligrams per deciliter, divide by 0.01129. To convert glucose values ​​to milligrams per deciliter, divide by 0.01129. To convert insulin values ​​to micrograms per liter, divide by 172.2. 【0076】 1. Fatty degeneration was assessed as the percentage of hepatocytes containing large and medium-sized intracytoplasmic lipid droplets (excluding foamy microvesicles) and graded according to the Fatty Degeneration, Activity, and Fibrosis (SAF) scoring system as 0 (<5%), 1 (5-33%), 2 (34-66%), or 3 (≧67%). Patients with grade 0 fatty degeneration were excluded from the study. 【0077】 2. Intralobular inflammation was classified according to the SAF scoring system as either Grade 1 (two small clusters of inflammatory cells) or Grade 2 (more than two clusters of inflammatory cells). 【0078】 3. Balloon-like degeneration was classified according to the Non-Alcoholic Steatohepatitis Clinical Research Network (NASH CRN) grading system as either Grade 1 (round hepatocytes with pale cytoplasm and similar size to normal hepatocytes) or Grade 2 (presence of hypertrophic hepatocytes with a diameter at least twice that of normal hepatocytes against a background of distinctly round hepatocytes). 【0079】 4. Fibrosis was classified according to the SAF-NASH CRN staging system as stage F0 (no fibrosis), stage F1 (mild fibrosis), stage F2 (significant [moderate] fibrosis), stage F3 (progressive fibrosis), or stage F4 (cirrhosis). Patients with stage F4 fibrosis were excluded from the study. 【0080】 5. The SAF-activity (SAF-A) score ranges from 0 to 4, with higher scores indicating more severe disease activity. 【0081】 6. The Non-Alcoholic Fatty Liver Disease Activity Score (NAS) ranges from 0 to 8. A score of 2 or less indicates "not NASH," a score of 3 or 4 indicates "borderline NASH," and a score of 5 to 8 indicates "definite NASH." 【0082】 7. The ABC3D score includes: A = Age > 50 years, B = BMI > 30 kg / m² 2 , C=platelet count<200×10 9 / L, 3 = PRO-C3 > 15.5 ng / mL, D = Diabetes mellitus = Present. The presence of each factor adds 1 point, except for T2DM which adds 2 points, for a maximum of 6 (see Boyle M et al, "Performance of the PRO-C3 collagen neo-epitope biomarker in non-alcoholic fatty liver disease", JHEP Rep. 2019;1(3):188-198). 【0083】 8.2 The FIBC3 score is calculated using the following formula: -5.939 + (0.053 × age (years)) + (0.076 × BMI (kg / m²)) 2 )) + (1.614 × T2DM (yes = 1, no = 0)) - (0.009 × platelets (10 9 / L))+(0.071×PRO-C3(ng / mL)) (see Boyle M et al,. “Performance of the PRO-C3 collagen neo-epitope biomarker in non-alcoholic fatty liver disease”, JHEP Rep. 2019;1(3):188-198). 【0084】 9 3 The FIB4 score is calculated using the following formula: (Age (years) × AST (U / L)) / (Platelet count (× 10) 9 / L)×√ALT(U / L)) (see Sterling RK, et al. “Development of a simple noninvasive index to predict significant fibrosis in patients with HIV / HCV coinfection”. Hepatology 2006;43(6):1317-25). 【0085】 The MACK3 score is based on the following parameters: AST (IU / L), blood glucose (mmol / L), insulin (μU / mL), and cytokeratin M30 (IU / L) (see Boursier J et al., “Screening for therapeutic trials and treatment indication in clinical practice: MACK-3, a new blood test for the diagnosis of fibrotic NASH. Aliment”, Pharmacol Ther. 2018;47(10):1387-9). 【0086】 11 5 The ELF score is calculated using the following formula: 2.494 + 0.846 ln (hyaluronic acid) + 0.735 ln (PIIINP) + 0.391 ln (TIMP-1) (see Lichtinghagen R et al. “The Enhanced Liver Fibrosis (ELF) score: normal values, influence factors and proposed cut-off values”, J Hepatol. 2013;59(2):236-42). 【0087】 12 6 The NAFLD fibrosis score is calculated using the following formula: -1.675 + 0.037 × age (years) + 0.094 × BMI (kg / m²) 2 ) + 1.13 × (Fasting blood glucose impairment / diabetes (yes=1, no=0)) + 0.99 × (AST / ALT ratio (unitless)) - 0.013 × platelets (× 10 9 / L)-0.66 × albumin (g / dL) (See Angulo Pet al. "The NAFLD fibrosis score: a non-invasive system that identifies liver fibrosis in patients with NAFLD", Hepatology 2007;45(4):846-54). 【0088】 The mean age of the patients was 55 years, and the mean body mass index (BMI) was 33. 89 patients (63%) were female, and 60 patients (42%) had type 2 diabetes. Significant or progressive fibrosis (stages F2 and F3, respectively) was present in 111 patients (78%), and most patients had highly active NASH (mean [±SD] SAF-A score was 3.3±0.5, with 73% having NAS≧6, indicating high disease activity). 【0089】 In the pooled ranifibranol group, 42 patients (30%) achieved resolution of NASH and improvement of liver fibrosis (≥stage 1 (E1)) [17 patients (24%) treated with 800 mg and 25 patients (35%) treated with 1200 mg]. 【0090】 Available diagnostic scores As a first step, we evaluated the performance of currently available diagnostic scores FIB4, FIBC3, ABC3D, NFS, ELF, and MACK3 in predicting histological responders with respect to endpoint E1. For MACK3, baseline values ​​and absolute changes between baseline and EOT were independently associated with E1. The resulting model showed an AUROC of 0.76 in the E1 assessment. For the other five scores, it was not possible to develop models because their results (baseline values, absolute changes, and relative changes) were not independently associated with E1. 【0091】 Table 3 shows the accuracy of the published diagnostic scores for predicting E1. 【0092】 [Table 3] 【0093】 No models achieved an AUROC score of 0.80 for E1, indicating that existing scores do not have sufficient accuracy to predict this endpoint. Therefore, further analysis focused on developing novel specific signatures for E1. 【0094】 Example 3 Development of a specific, non-invasive biomarker signature for E1 (disappearance of NASH and improvement of fibrosis ≥ Stage 1). Signatures were constructed for each of the 65 available test biomarkers in the study, considering three different methods (baseline raw value, absolute change in EOT, and relative change in EOT), resulting in a total of 195 biomarkers. Univariate analysis, PCA, and sPLS-DA yielded a first shortlist of 52 biomarkers associated with endpoint E1. Of these, 10 biomarkers were selected for the final shortlist (*) (see Table 4). 【0095】 [Table 4] 【0096】 Of these 10 biomarkers, four (baseline adiponectin and ferritin, and relative changes in MMP9 and transferrin) were ultimately selected as independent predictors of E1 using logistic regression. By combining these four parameters to obtain an E1 score, an AUROC of 0.81 ± 0.08 was obtained for predicting the E1 response. Calibration of E1 was good, with a Brier score of 0.17 (see Table 5). 【0097】 [Table 5] 【0098】 The curves for sensitivity (descending curve, top left to bottom right), specificity (rising curve, bottom left to top right), NPV (descending curve, top left to center / top right), and PPV (rising curve, bottom left / center to top right) are shown in Figure 1 as functions of the E1 score results. The calculated thresholds for 80% NPV and 80% PPV were 0.299 and 0.592, respectively. Sensitivity was 70% at the 80% NPV threshold, and specificity was 95% at the 80% PPV threshold. Using these two thresholds, 54% of patients were diagnosed as non-responders, 18% as responders, and 28% were placed in the gray zone of uncertain diagnosis. Ultimately, based on the cutoff values ​​of the 80% positive and negative predictive values ​​for the signature, 72% of the population was classified as responders or non-responders, of which 81% could be histologically confirmed (non-invasive diagnostic accuracy). 【0099】 All of these results suggest that the derived signature can be considered a good classification indicator for endpoint E1. 【0100】 Thus, this specific combination of biomarkers (adiponectin, ferritin, MMP9, and transferrin) enabled the evaluation of the effectiveness of ranifibranol treatment in non-cirrhotic NASH, and the results showed good diagnostic performance, particularly regarding the resolution of NASH and improvement of fibrosis ≥1 stage. 【0101】 Aspects of this disclosure will be further described with reference to the following non-limiting embodiments. 1. A method for evaluating the effectiveness of treatment with ranifibranol in patients with liver disease, a) To measure in vitro the levels of a combination of biomarkers consisting of adiponectin, ferritin, MMP9, and transferrin in biological samples obtained from the above patients; b) Evaluate the effectiveness of treatment with ranifibranol in the above patients according to the measured levels of the above biomarker combinations. A method that includes this. 【0102】 2. A method for evaluating the effectiveness of treatment with ranifibranol in patients with liver disease, a1) In vitro measurement of the levels of a combination of biomarkers consisting of adiponectin, ferritin, MMP9, and transferrin in biological samples obtained from the above patients; b1) Comparing the levels measured in step a) with the levels measured in multiple samples from liver disease patients who have received treatment with ranifibranol and whose treatment efficacy is known, wherein the comparison is performed by a statistical learning model that uses the levels of the combination of biomarkers measured in step a) as input data; c1) Evaluate the effectiveness of treatment with ranifibranol in the above patients according to the results determined by the model specified in step b1) A method that includes this. 【0103】 3. The method according to item 1 or 2, wherein adiponectin and ferritin levels are measured before the initiation of treatment with ranifibranol, and MMP9 and transferrin levels are measured before the initiation of treatment and after at least three months of treatment with ranifibranol. 【0104】 4. The method according to item 3, wherein the levels of the combination of biomarkers measured in step a1) are used to obtain a score related to the evaluation of the effectiveness of the treatment in the patient, and the score is compared to at least one predetermined cutoff value to classify the prognosis into several classes. 【0105】 5. The method according to claim 4, wherein the above-mentioned classes include at least two classes, one of which is a class that does not respond to treatment with ranifibranol. 【0106】 6. The method of paragraph 4 or 5, wherein the effectiveness of the treatment in the above patient is evaluated by comparing the above score with a first calculated cutoff value, below which the effectiveness is predicted to be poor or nonexistent, and a second calculated cutoff value, above which the effectiveness is predicted to be good. 【0107】 7. The method according to any one of items 2 to 6, wherein the learning model described above is based on a prior analysis of samples from a cohort including patients who show a good response to treatment with ranifibranol and patients who show a poor or no response to treatment with ranifibranol. 【0108】 8. The method according to paragraph 7, wherein the above pre-analysis includes the application of learning and variable selection methods. 【0109】 9. The method described in item 8, wherein the learning and variable selection method is logistic regression. 【0110】 10. The method according to any of items 7 to 9, wherein the above levels are weighted according to the prior analysis of the above cohort to derive the above score. 【0111】 11. The method according to item 1 or 2, wherein the biological sample is a biological fluid sample. 【0112】 12. The method according to item 11, wherein the sample of biological fluid is a sample of blood, serum, or plasma. 【0113】 13. The method according to item 1 or 2, wherein the biomarker whose level is measured in step a) is a protein biomarker. 【0114】 14. The method according to item 1 or 2, wherein the liver disease is non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, compensated or decompensated cirrhosis, hepatic fibrosis, fatty liver disease, acute liver failure, or acute exacerbation of chronic liver failure. 【0115】 15. A system for evaluating the effectiveness of treatment with ranifibranol in patients with liver disease, a) Means for measuring the levels of a combination of biomarkers consisting of adiponectin, ferritin, MMP9, and transferrin in a biological sample obtained from the above patient, or for receiving such measurement data; b) Measurement data processing means configured to evaluate the effectiveness of treatment with ranifibranol in the patient according to the measured levels of the above combination of biomarkers. A system that includes this. 【0116】 16. A combination of biomarkers consisting of adiponectin, ferritin, MMP9, and transferrin for use in evaluating the efficacy of ranifibranol treatment in patients with liver disease. 【0117】 While the subject matter and its merits have been described in detail, it should be understood that various changes, substitutions, and modifications may be made herein without departing from the spirit and scope of the application as defined by the appended claims. Furthermore, the scope of the application is not intended to be limited to specific embodiments of the processes, material compositions, means, methods, and steps described herein. As will be readily apparent to those skilled in the art from the disclosure of the subject matter, existing or subsequently developed processes, material compositions, means, methods, or steps that perform substantially the same function or achieve substantially the same results as the corresponding embodiments described herein may be used in accordance with the subject matter of this disclosure. Accordingly, the appended claims are intended to include within their scope such processes, material compositions, means, methods, or steps. 【0118】 In addition to the various embodiments illustrated and claimed, the subject matter of this disclosure also relates to other embodiments having any other possible combination of the features disclosed and claimed herein. Thus, the subject matter of this disclosure includes any suitable combination of the features disclosed herein, as the specific features presented herein can be combined with one another in other ways within the scope of the subject matter of this disclosure. In this manner, the above description of specific embodiments of the subject matter of this disclosure is presented for illustrative and explanatory purposes only. It is not intended to be exhaustive or to limit the subject matter of this disclosure to the embodiments disclosed herein. 【0119】 Those skilled in the art will see that various modifications and alterations can be made to the apparatus, methods and systems of the subject matter of this disclosure without departing from the spirit or scope of the subject matter of this disclosure. Accordingly, the subject matter of this disclosure is intended to include modifications and alterations that fall within the scope of the appended claims and their equivalents. 【0120】 All patents, patent applications, publications, product descriptions, and protocols cited throughout this application are incorporated herein by reference in their entirety for all purposes.

Claims

[Claim 1] A method for evaluating the effectiveness of treatment with ranifibranol in patients with liver disease, a) In vitro measurement of the levels of a combination of biomarkers consisting of adiponectin, ferritin, MMP9, and transferrin in biological samples obtained from the above patients; b) Evaluate the effectiveness of treatment with ranifibranol according to the level measured in step a) and A method that includes this. [Claim 2] The method according to claim 1, wherein adiponectin and ferritin levels are measured before the initiation of treatment with ranifibranol. [Claim 3] The method according to claim 1 or 2, wherein MMP9 and transferrin levels are measured before the initiation of treatment with ranifibranol and after at least three months of treatment with ranifibranol. [Claim 4] The method according to claim 1 or 2, wherein the level measured in step a) is used to obtain a score to which the result belongs to a confirmed or uncertain prognosis class. [Claim 5] The method according to claim 4, wherein the above score is obtained by combining the levels measured in step a) in a mathematical function. [Claim 6] The method according to claim 5, wherein the above mathematical function is a binomial logistic regression. [Claim 7] The method according to claim 4, comprising comparing the score with a first calculated cutoff value, which, if below the cutoff value, predicts non-response to ranifibranol treatment. [Claim 8] The method according to claim 4, comprising comparing the score with a second calculated cutoff value, which, if exceeded, predicts a response to ranifibranol treatment. [Claim 9] The method according to claim 1 or claim 2, wherein the above-mentioned biological sample is a sample of biological fluid. [Claim 10] The method according to claim 9, wherein the biological fluid is blood, serum, or plasma. [Claim 11] The method according to claim 1 or claim 2, wherein the liver disease is non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, compensated or decompensated cirrhosis, hepatic fibrosis, fatty liver disease, acute liver failure, or acute exacerbation of chronic liver failure. [Claim 12] A system for evaluating the effectiveness of treatment with ranifibranol in patients with liver disease, a) Means for measuring the levels of a combination of biomarkers consisting of adiponectin, ferritin, MMP9, and transferrin in a biological sample obtained from the above patient, or for receiving such measurement data; b) A data processing means configured to evaluate the effectiveness of treatment with ranifibranol in the patient according to the measured levels of the combination of biomarkers described above. A system that includes this. [Claim 13] A combination of biomarkers consisting of adiponectin, ferritin, MMP9, and transferrin for use in evaluating the efficacy of ranifibranol treatment in patients with liver disease. [Claim 14] The combination according to claim 13, wherein the liver disease is non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, compensated or decompensated cirrhosis, hepatic fibrosis, fatty liver disease, acute liver failure, or acute exacerbation of chronic liver failure. [Claim 15] A method for treating liver disease in the subject, a) In vitro measurement of the levels of a combination of biomarkers consisting of adiponectin, ferritin, MMP9, and transferrin in biological samples obtained from the above patients before the start of treatment; b) Administer an effective dose of ranifibranol daily to the above subjects for at least three months; c) Measuring the levels of MMP9 and transferrin in the above-mentioned biological samples in vitro after at least three months of treatment; d) Evaluate the effectiveness of treatment with ranifibranol according to the levels measured in steps a) and c), thereby predicting a response or non-response to ranifibranol treatment; e) If the evaluation performed in step d) predicts the response to ranifibranol treatment, or if a diagnosis cannot be made, the subject shall continue to receive an effective dose of ranifibranol for at least another three months. A method that includes this. [Claim 16] The method according to claim 15, wherein the evaluation in step d) is performed by obtaining a score from the levels measured in steps a) and c), and comparing the score with (i) a first calculated cutoff value, below which non-response to ranifibranol treatment is predicted, and (ii) a second calculated cutoff value, above which a response to ranifibranol treatment is predicted. [Claim 17] The method according to claim 16, wherein if the acquired score exceeds the second calculation cutoff value described above, it is predicted that NASH will disappear and liver fibrosis will improve by one stage or more. [Claim 18] The method according to any one of claims 15 to 17, wherein the above-mentioned biological sample is a sample of biological fluid. [Claim 19] The method according to claim 18, wherein the biological fluid is blood, serum, or plasma. [Claim 20] The method according to any one of claims 15 to 17, wherein the liver disease is non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, compensated or decompensated cirrhosis, hepatic fibrosis, fatty liver disease, acute liver failure, or acute exacerbation of chronic liver failure.

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