Methods for diagnosing and evaluating non-alcoholic steatohepatitis

Abstract

The invention relates to a method for the diagnosis of non-alcoholic steatohepatitis (NASH), for determining the activity, the stage, or the severity of NASH or for classifying a subject as a potential receiver or non receiver of a treatment of NASH using circulating miRNAs and other blood circulating markers of liver damage, e.g. alpha 2 macroglobulin, HbA1c, N-terminal pro-peptide of collagen type III, miR-34 and miR-200. It also relates to a kit for implementing the method of the invention, and the compounds for use in a method for the treatment of NASH, wherein the subject to be treated is identified, evaluated or classified according to the method of the invention.

1 - 16 . (canceled) 
     
     
         17 . A method for the diagnosis of non-alcoholic steatohepatitis (NASH) or for determining the activity, the stage, or the severity of NASH in a subject, comprising the steps of:
 (a) providing blood samples from said subject,   (b) separating a cell-free, platelet free plasma sample from one of said blood sample,   (c) extracting total RNA from said cell-free, platelet free plasma sample, and   (d) measuring in one of said blood samples obtained in step (a) the level(s) of one or more markers of (i) alpha 2 macroglobulin, (ii) glycated haemoglobin (HbA1c) and (iii) N-terminal pro-peptide of collagen type III, each of which is measured by an antibody specific to it, and   (e) measuring in total RNA extracted in step (c) one or both markers of (iv) hsa-miR-34a-5p and (v) hsa-miR-200a-3p, each of which is measured using a nucleic acid specific to SEQ ID NO:3 or SEQ ID NO:4.   
     
     
         18 . The method according to  claim 17 , wherein the separation of a cell-free, platelet free plasma sample from one of the blood samples of said subject comprises the following steps:
 adding the blood sample in a citrate containing tube,   centrifugating said blood sample at 1,500×g for collecting the supernatant containing the cell-free plasma, and   centrifugating said cell-free plasma at 13,000×g for collecting the supernatant containing the cell-free, platelet free plasma sample.   
     
     
         19 . The method according to  claim 17 , comprising the implementation of the following measures:
 (i) determining the level of alpha 2 macroglobulin;   (ii) determining the level of HbA1c;   (iii) determining the level of N-terminal pro-peptide of collagen type III;   (iv) determining the level of hsa-miR-34a-5p; and   (v) determining the level of hsa-miR-200a-3p;   
       wherein an increase in said levels relative to that of a control sample is indicative of NASH and/or of the activity, the stage, or the severity of NASH. 
     
     
         20 . The method according to  claim 17 , wherein a NASH score is calculated from the levels determined in measures (i) to (v) and wherein NASH is diagnosed and/or NASH stage or activity or severity is determined on the basis of said NASH score. 
     
     
         21 . The method according to  claim 20 , wherein a NASH score is calculated according to the following logistic function: 
       
         
           
             
               S 
               = 
               
                 
                   e 
                   Y 
                 
                 
                   1 
                   + 
                   
                     e 
                     Y 
                   
                 
               
             
           
         
         wherein:
     Y=k+a*A+b*B+c*C+d*D+f*F    
 
         wherein: 
         S is the NASH score; 
         A is the level of alpha 2 macroglobulin in g/L; 
         B is the level of HbA1c in percent; 
         C is the level of N-terminal pro-peptide of collagen type III in ng/mL; 
         D is the level of hsa-miR-34a-5p in Cq; 
         F is the level of hsa-miR-200a-3p in Cq; 
         k is a number comprised between 5.26 and 36.76; 
         a is a number comprised between 0.11 and 1.07; 
         b is a number comprised between 0 and 1.02; 
         c is a number comprised between 0 and 0.23; 
         d is a number comprised between −0.77 and 0; 
         f is a number comprised between −0.78 and 0. 
       
     
     
         22 . The method according to  claim 21 , wherein a NASH score higher than a threshold value comprised between 0.102 and 0.6465 is indicative of a severe NASH, or of a moderate or high NASH activity. 
     
     
         23 . A method for classifying a subject as a receiver or non receiver of a treatment for non-alcoholic steatohepatitis (NASH), comprising the steps of:
 (a) providing blood samples from said subject,   (b) separating a cell-free, platelet free plasma sample from one of said blood samples,   (c) extracting total RNA from said cell-free, platelet free plasma sample, and   (d) measuring in one of said blood samples obtained in step (a) the level(s) of one or more markers of (i) alpha 2 macroglobulin, (ii) glycated haemoglobin (HbA1c) and (iii) N-terminal pro-peptide of collagen type III, each of which is measured by an antibody specific to it,   and(e) measuring in total RNA extracted in (c) one or both markers of (iv) hsa-miR-34a-5p and (v) hsa-miR-200a-3p, each of which is measured using a nucleic acid specific to SEQ ID NO:3 or SEQ ID NO:4.   
     
     
         24 . The method according to  claim 23 , comprising the implementation of the following measures:
 (i) determining the level of alpha 2 macroglobulin;   (ii) determining the level of HbA1c;   (iii) determining the level of N-terminal pro-peptide of collagen type III;   (iv) determining the level of hsa-miR-34a-5p; and   (v) determining the level of hsa-miR-200;   
       wherein a NASH score is calculated from the levels determined in measures (i) to (v) and wherein the subject is classified as a receiver or non receiver of the treatment for NASH based on said score. 
     
     
         25 . The method according to  claim 23 , comprising determining the level of alpha 2 macroglobulin, HbA1c, N-terminal pro-peptide of collagen type III, hsa-miR-34a-5p and hsa-miR-200a-3p wherein the NASH score is calculated according to the following logistic function: 
       
         
           
             
               S 
               = 
               
                 
                   e 
                   Y 
                 
                 
                   1 
                   + 
                   
                     e 
                     Y 
                   
                 
               
             
           
         
         wherein:
     Y=k+a*A+b*B+c*C+d*D+f*F    
 
         wherein: 
         S is the NASH score; 
         A is the level of alpha 2 macroglobulin in g/L; 
         B is the level of HbA1c in percent; 
         C is the level of N-terminal pro-peptide of collagen type III in ng/mL; 
         D is the level of hsa-miR-34a-5p in Cq; 
         F is the level of hsa-miR-200a-3p in Cq; 
         k is a number comprised between 5.26 and 36.76; 
         a is a number comprised between 0.11 and 1.07; 
         b is a number comprised between 0 and 1.02; 
         c is a number comprised between 0 and 0.23; 
         d is a number comprised between −0.77 and 0; 
         f is a number comprised between −0.78 and 0. 
       
     
     
         26 . The method according to  claim 25 , wherein the subject is classified as a receiver of the treatment if S is higher than a threshold value comprised between 0.102 and 0.6465. 
     
     
         27 . A method for calculating a NASH score which is indicative of non-alcoholic steatohepatitis (NASH), or of the activity, the stage, or the severity of NASH in a subject, wherein said score is based on the steps of:
 (a) providing blood samples from said subject;   (b) separating a cell-free, platelet free plasma sample from one of said blood samples,   (c) extracting total RNA from said cell-free, platelet free plasma sample, and   (d) measuring in one of said blood samples obtained in step (a) the level(s) of one or more markers of (i) alpha 2 macroglobulin, (ii) glycated haemoglobin (HbA1c) and (iii) N-terminal pro-peptide of collagen type III, each of which is measured by an antibody specific to it, and (e) measuring in total RNA extracted in (c) one or both markers of (iv) hsa-miR-34a-5p or (v) hsa-miR-200a-3p, each of which is measured using a nucleic acid specific to SEQ ID NO:3 or SEQ ID NO:4.   
     
     
         28 . The method of  claim 27 , comprising determining the level of alpha 2 macroglobulin, HbA1c, N-terminal pro-peptide of collagen type III, hsa-miR-34a-5p and hsa-miR-200a-3p, wherein the NASH score is calculated according to the following logistic function: 
       
         
           
             
               S 
               = 
               
                 
                   e 
                   Y 
                 
                 
                   1 
                   + 
                   
                     e 
                     Y 
                   
                 
               
             
           
         
         wherein:
     Y=k+a*A+b*B+c*C+d*D+f*F    
 
         wherein: 
         S is the NASH score; 
         A is the level of alpha 2 macroglobulin in g/L; 
         B is the level of HbA1c in percent; 
         C is the level of N-terminal pro-peptide of collagen type III in ng/mL; 
         D is the level of hsa-miR-34a-5p in Cq; 
         F is the level of hsa-miR-200a-3p in Cq; 
         k is a number comprised between 5.26 and 36.76; 
         a is a number comprised between 0.11 and 1.07; 
         b is a number comprised between 0 and 1.02; 
         c is a number comprised between 0 and 0.23; 
         d is a number comprised between −0.77 and 0; 
         f is a number comprised between −0.78 and 0. 
       
     
     
         29 . A kit comprising means for determining the level of:
 (i) alpha 2 macroglobulin;   (ii) at least one marker selected in the group consisting of HbA1c, fasting glucose level, fructosamine level;   (iii) N-terminal pro-peptide of collagen type III;   (iv) at least one marker selected in the group consisting of has-miR-34a-5p; and   (v) hsa-miR-200a-3p.   
     
     
         30 . The method according to  claim 23 , wherein the separation of a cell-free, platelet free plasma sample from one of the blood samples of said subject comprises the following steps:
 adding the blood sample in a citrate containing tube,   centrifugating said blood sample at 1,500×g for collecting the supernatant containing the cell-free plasma, and   centrifugating said cell-free plasma at 13,000×g for collecting the supernatant containing the cell-free, platelet free plasma sample.   
     
     
         31 . The method according to  claim 27 , wherein the separation of a cell-free, platelet free plasma sample from one of the blood samples of said subject comprises the following steps:
 adding the blood sample in a citrate containing tube,   centrifugating said blood sample at 1,500×g for collecting the supernatant containing the cell-free plasma, and   centrifugating said cell-free plasma at 13,000×g for collecting the supernatant containing the cell-free, platelet free plasma sample.   
     
     
         32 . A method for treating non-alcoholic steatohepatitis (NASH) in a subject, comprising:
 (a) diagnosing NASH or determining the activity, the stage, or the severity of NASH in a subject according to the method of  claim 17 ;   (b) administering a treatment for NASH to said subject based on step (a),   wherein the treatment comprises administering to the subject a compound of formula (I):   
       
         
           
           
               
               
           
         
         wherein: 
         X1 represents a halogen, a R1, or G1-R1 group; 
         A represents a CH═CH or a CH2-CH2 group; 
         X2 represents a G2-R2 group; 
         G1 represents an atom of oxygen; 
         G2 represents an atom of oxygen or sulfur; 
         R1 represents a hydrogen atom, an unsubstituted alkyl group, an aryl group or an alkyl group that is substituted by one or more halogen atoms, an alkoxy or an alkylthio group, cycloalkyl groups, cycloalkylthio groups or heterocyclic groups; 
         R2 represents an alkyl group substituted by at least a —COOR3 group, wherein R3 represents a hydrogen atom, or an alkyl group that is substituted or not by one or more halogen atoms, cycloalkyl groups, or heterocyclic groups; 
         R4 and R5, identical or different, representing an alkyl group that is substituted or not by one or more halogen atoms, cycloalkyl groups, heterocyclic groups; 
         or a pharmaceutically acceptable salt thereof. 
       
     
     
         33 . A method for treating non-alcoholic steatohepatitis (NASH) in a subject, comprising:
 (a) classifying said subject as a receiver or non-receiver of a treatment for non-alcoholic steatohepatitis (NASH) according to the method of  claim 23 ;   (b) administering a treatment for NASH to said subject based on step (a),   wherein the treatment comprises administering to the subject a compound of formula (I):   
       
         
           
           
               
               
           
         
         wherein: 
         X1 represents a halogen, a R1, or G1-R1 group; 
         A represents a CH═CH or a CH2-CH2 group; 
         X2 represents a G2-R2 group; 
         G1 represents an atom of oxygen; 
         G2 represents an atom of oxygen or sulfur; 
         R1 represents a hydrogen atom, an unsubstituted alkyl group, an aryl group or an alkyl group that is substituted by one or more halogen atoms, an alkoxy or an alkylthio group, cycloalkyl groups, cycloalkylthio groups or heterocyclic groups; 
         R2 represents an alkyl group substituted by at least a —COOR3 group, wherein R3 represents a hydrogen atom, or an alkyl group that is substituted or not by one or more halogen atoms, cycloalkyl groups, or heterocyclic groups; 
         R4 and R5, identical or different, representing an alkyl group that is substituted or not by one or more halogen atoms, cycloalkyl groups, heterocyclic groups; 
         or a pharmaceutically acceptable salt thereof.