KR102573089B1 - Novel Biomarkers for Predicting Susceptibility to nonalcoholic fatty liver disease treatment agent and Uses Thereof - Google Patents

Abstract

Using the biomarker for predicting susceptibility to OCA, a non-alcoholic fatty liver treatment agent of the present invention, it is possible to reliably determine the susceptibility of each patient before starting treatment, thereby minimizing unnecessary drug administration and increasing treatment efficiency. .

Description

Biomarkers for Predicting Susceptibility to nonalcoholic fatty liver disease treatment agent and Uses Thereof

The present invention relates to a biomarker for predicting susceptibility to non-alcoholic fatty liver treatment and its use.

Fatty liver disease (fatty liver disease), also called fatty liver, is a disease that causes liver failure due to abnormal accumulation of fat (triglyceride, etc.) in hepatocytes. It is known that the initial condition of fatty liver disease is simple fatty liver in which only fat deposition is recognized in hepatocytes, and then the condition progresses to steatohepatitis (including liver fibrosis), further cirrhosis, or hepatocellular carcinoma. In general, causes of fat deposition in the liver include alcohol consumption, obesity, diabetes, abnormal lipid metabolism, drugs (steroids, tetracycline, etc.), Cushing's syndrome, addiction (yellow phosphorus, etc.), and severe nutritional disorders. there is.

The causes of fatty liver disease are largely divided into alcoholic and non-alcoholic. Liver disease caused by the former is alcoholic fatty liver disease (also called alcoholic liver disease), and liver disease caused by the latter is non-alcoholic fatty liver disease ( It is also called nonalcoholic fatty liver disease (NAFLD).

Non-alcoholic fatty liver disease is defined as fatty change (steatosis), lobular hepatitis, and fatty change (steatosis), which are characteristic findings of alcoholic hepatitis in a liver biopsy, despite the absence of a history of alcohol intake that is recognized as harmful to the liver. steatohepatitis). Pathological findings of the liver show a diverse spectrum from simple fatty liver (Non-Alcoholic Fatty Liver, NAFL) to Non-Alcoholic Steatohepatits (NASH), fibrosis accompanied by steatohepatitis, and cirrhosis. It is used in the sense of including all.

Most of these non-alcoholic fatty liver diseases are accompanied by insulin resistance, obesity, diabetes, and hyperlipidemia. When such a complication exists, it is necessary to treat it first. The principle of treatment for non-alcoholic fatty liver disease is lifestyle improvement such as diet therapy and exercise therapy, but it is difficult to reliably implement it in reality. In non-alcoholic steatohepatitis, since the possibility of progressing to cirrhosis hepatocellular carcinoma is high, more aggressive drug treatment is required. Treatments aimed at improving oxidative stress and insulin resistance, which are considered important for the pathological development of non-alcoholic steatohepatitis, have also been attempted, but the reality is that there is no treatment for which sufficient scientific evidence has been established.

Currently, obeticholic acid (OCA) is in clinical progress as the only drug for the treatment of non-alcoholic fatty liver disease, which has a high prevalence due to westernized eating habits worldwide, but OCA is known to show a low treatment response rate of 23%. . Accordingly, it is necessary to develop a method capable of selecting a patient group expected to have a high response to OCA treatment, minimizing unnecessary drug administration, and monitoring the treatment response during drug treatment.

Patent Publication No. 10-2017-0095271

Under this circumstance, the inventors of the present invention identified the causes of different susceptibility to Obeticholic acid (OCA), a non-alcoholic fatty liver treatment, in non-alcoholic fatty liver patients, and developed a method for predicting the sensitivity, Obeticholic acid (OCA) As a biomarker predicting susceptibility in non-alcoholic fatty liver, the activity of the Cyp7b1 (Cytochrome P450 Family 7 Subfamily B Member 1) gene and / or its protein was analyzed, and as a result, the Cyp7b1 gene and / or protein in liver tissue The present invention was completed by confirming that the degree of inhibition of liver fibrosis due to the drug sensitivity of obeticholic acid (OCA) is different depending on the activity aspect of.

Accordingly, an object of the present invention is to provide a biomarker for predicting sensitivity to obeticholic acid (OCA), a non-alcoholic fatty liver treatment agent.

In addition, another object of the present invention is to provide a composition for predicting sensitivity to obeticholic acid (OCA), a non-alcoholic fatty liver treatment agent.

In addition, another object of the present invention is to provide a kit for predicting sensitivity to obeticholic acid (OCA), a non-alcoholic fatty liver treatment agent.

In addition, another object of the present invention is to provide a method for providing information for predicting sensitivity to obeticholic acid (OCA), a non-alcoholic fatty liver treatment agent.

Hereinafter, the present invention will be described in more detail.

According to one aspect of the present invention, the present invention Cyp7b1 (Cytochrome P450 Family 7 Subfamily B Member 1) gene; Or it provides a biomarker or biomarker composition for predicting susceptibility to obeticholic acid (OCA), a non-alcoholic fatty liver treatment comprising a protein thereof.

The present invention is a Cyp8b1 (Cytochrome P450 Family 8 Subfamily B Member 1) gene as a biomarker for predicting sensitivity to obeticholic acid (OCA), a non-alcoholic fatty liver treatment; Or it may further include a protein thereof.

The greatest feature of the present invention is to predict susceptibility to OCA by using the activity of the Cyp7b1 and/or Cyp8b1 gene and its product, the active protein, as a biomarker.

The biomarker of the present invention can be a sensitivity indicator for OCA, a non-alcoholic fatty liver treatment for non-alcoholic fatty liver patients, and can be used for non-alcoholic fatty liver treatment because it has excellent accuracy and reliability as a sensitivity marker for OCA. .

As used herein, the term "susceptibility" means whether a specific drug for the treatment of non-alcoholic fatty liver of an individual patient exhibits an effect.

The term "sensitivity" used herein while referring to sensitivity means that the efficacy of a drug works by reacting sensitively to a corresponding drug, and is used interchangeably with sensitivity within the present specification.

The term "resistance" used herein while referring to sensitivity means not reacting sensitively to a corresponding drug so that the efficacy of the drug does not work.

For example, the OCA may or may not show an effect depending on non-alcoholic fatty liver patients. Whether or not OCA exhibits an effect on non-alcoholic fatty liver of such an individual patient is referred to as sensitivity. Therefore, if it is possible to predict patients for whom an effect can be expected (responders) and patients for whom no effect can be expected (non-responders) before the start of treatment according to the present invention, chemotherapy with high efficacy and safety can be realized.

As used herein, the term "prediction" is used herein to refer to the likelihood that a subject patient will respond favorably or unfavorably to a drug or set of drugs. In one embodiment, the prediction relates to the extent of this response.

According to a preferred embodiment of the present invention, the biomarker of the present invention may further include other genes and products included in the biosynthetic pathway associated with the Cyp7b1 gene or its protein. Specifically, Cyp7b1 is known to be involved in the acidic pathway producing CDCA (Chenodeoxycholic acid) as an end product among bile acid synthesis pathways. Accordingly, the gene involved in the acidic pathway and the end product CDCA may be further included as biomarkers.

In the present invention, when the expression level of the Cyp7b1 gene or its protein expression or activity level, which is the present biomarker, is higher than normal, it is determined that there is sensitivity to OCA; When the expression level of the Cyp7b1 gene or the expression or activity level of its protein is lower than normal, it can be determined that there is resistance to OCA.

In the present invention, as the expression level of the Cyp7b1 gene or its protein expression or activity level is higher than the expression level of the Cyp8b1 gene or its protein expression or activity level, the sensitivity to OCA is higher. Specifically, Cyp7b1:Cyp8b1 When the ratio is 5 or more, preferably 6 or more, it can be determined that the sensitivity to OCA is high. Here, Cyp8b1 refers to one of the Cytochrom p450 familys (Cyp7a1, Cyp8b1, and Cyp27a1) that play a major role in generating bile acids by being involved in the neutral pathway.

According to another aspect of the present invention, the present invention is Cyp7b1 and / or Cyp8b1 gene expression level; Or it provides a composition for predicting susceptibility to OCA, including an agent for measuring the expression or activity level of its protein.

As used herein, the term "non-alcoholic fatty liver" refers to rectal cancer, colon cancer, and anal cancer.

Unless otherwise specified herein, the expression "expression level of a gene; or measurement of the protein expression or activity level of the gene" used herein means detecting a target to be detected in a corresponding sample.

In the present invention, an object to be detected is mRNA and/or protein of a corresponding gene in a sample. That is, it is possible to determine whether the gene is expressed by detecting RNA, which is a transcription product of a gene, or protein (preferably, an active form), which is a gene product.

Detection of RNA or protein can usually be performed by extracting RNA or protein from a sample and detecting RNA or protein in the extract. Detection of such RNA or protein may be measured by immunoassay methods, hybridization reactions, and amplification reactions, but is not limited thereto and can be easily performed using various techniques known in the art.

In addition, according to a preferred embodiment of the present invention, the agent for measuring the gene expression level includes an antisense oligonucleotide, a primer pair or a probe that specifically binds to the mRNA of the gene.

The agent for measuring the expression of the mRNA is selected from the group consisting of antisense oligonucleotides specific to the gene, primer pairs, probes, and combinations thereof. That is, detection of a nucleic acid may be performed by an amplification reaction using one or more oligonucleotide primers that hybridize to a nucleic acid molecule encoding a gene or a complement of the nucleic acid molecule.

For example, mRNA detection using primers can be performed by amplifying a gene sequence using an amplification method such as PCR, and then confirming whether the gene is amplified by a method known in the art.

In addition, according to a preferred embodiment of the present invention, the agent for measuring the expression or activity level of the protein includes an antibody, peptide or nucleotide that specifically binds to the protein.

An agent for measuring the expression or activity level of the protein refers to an antibody that specifically binds to the protein, and includes polyclonal antibodies, monoclonal antibodies, recombinant antibodies, and combinations thereof.

Such antibodies include polyclonal antibodies, monoclonal antibodies, recombinant antibodies and complete forms having two full-length light chains and two full-length heavy chains, as well as functional fragments of antibody molecules, such as Fab, F(ab' ), F(ab')2 and Fv. Antibody production can be easily prepared using techniques well known in the field to which the present invention pertains, and commercially available antibodies can be used.

The composition of the present invention further includes not only agents for measuring the expression of the above-described genes, but also labels that enable quantitative or qualitative measurement of the formation of antigen-antibody complexes, conventional tools and reagents used in immunological analysis, and the like. can include

Labels capable of qualitatively or quantitatively measuring the formation of antigen-antibody complexes include, but are not limited to, enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioisotopes. . Enzymes available as detection labels include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glucoseoxy Multiple drugs, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphophenolpyruvate decarboxylase, β- Latamase and the like, but are not limited thereto. Fluorescent substances include fluorescein, isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanine, o-phthalaldehyde, fluorescamine, and the like, but are not limited thereto. Ligands include biotin derivatives and the like, but are not limited thereto. Luminescent substances include acridinium ester, luciferin, luciferase, and the like, but are not limited thereto. Microparticles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K4W(CN)8, [Os(bpy)3]2+, [ RU(bpy)3]2+, [MO(CN)8]4-, etc., but are not limited thereto. Radioactive isotopes include, but are not limited to, 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, and 186Re.

Examples of such tools or reagents include, but are not limited to, suitable carriers, solubilizers, detergents, buffers, stabilizers, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. The carrier includes a soluble carrier and an insoluble carrier, and an example of the soluble carrier is a physiologically acceptable buffer known in the art, such as PBS, and an example of the insoluble carrier is polystyrene, polyethylene, polypropylene, polyester, poly acrylonitrile, fluororesins, cross-linked dextran, polysaccharides, other papers, glass, metals, agarose, and combinations thereof.

Since the composition of the present invention uses the aforementioned biomarkers, redundant descriptions thereof are omitted to avoid excessive complexity of the present specification.

According to another aspect of the present invention, the present invention provides a kit for predicting susceptibility to OCA, including the composition.

The kit includes expression of the gene; Alternatively, tools and reagents commonly used in the art used for immunological analysis may be included, as well as preparations for measuring the expression or activity level of the protein thereof.

Examples of such tools or reagents include, but are not limited to, suitable carriers, labeling substances capable of generating a detectable signal, chromophores, solubilizers, detergents, buffers, stabilizers, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. The carrier includes a soluble carrier and an insoluble carrier, and an example of the soluble carrier is a physiologically acceptable buffer known in the art, such as PBS, and an example of the insoluble carrier is polystyrene, polyethylene, polypropylene, polyester, poly polymers such as acrylonitrile, fluororesin, crosslinked dextran, polysaccharide, latex-coated magnetic microparticles, other paper, glass, metal, agarose, and combinations thereof.

Since the kit of the present invention includes the aforementioned biomarkers and compositions, redundant descriptions thereof are omitted to avoid excessive complexity of the present specification.

According to another aspect of the present invention, the present invention

(a) preparing a biological sample from the subject;

(b) the expression level of the Cyp7b1 gene in the biological sample; Or measuring the expression or activity level of the protein of the gene; and

(c) determining the susceptibility of the subject to OCA based on the result of checking the level measured in step (b);

Provides an information providing method for predicting susceptibility to OCA, including.

The method of the present invention obtains a biological sample from a subject patient, measures the expression of one or a plurality of genes selected from the group consisting of the above-described genes in the sample, compares the expression with a normal sample, and or when the expression or activity of the protein is suppressed or reduced, determining that the sample is susceptible to OCA; and a step of judging that the sample is resistant to OCA when the expression or activity of the described gene or protein is increased or improved.

The method of the present invention is characterized in that the expression (preferably, active form) of a specific gene or protein in a sample is used as an indicator of sensitivity to OCA.

According to a preferred embodiment of the present invention, the expression level of the Cyp7b1 gene in step (c); Or, if the expression or activity level of the protein is higher than normal or high, it is determined that the subject has sensitivity (sensitivity) to COA.

According to a preferred embodiment of the present invention, the expression level of the Cyp7b1 gene in step (c); Or, if the expression or activity level of its protein is low or low compared to normal, it is determined that the subject has resistance to OCA.

As used herein, the term "low expression" or "low activity" when referring to the expression level of the genes means that a biomarker indicates or is indicative of an abnormal process, disease, or other condition in an individual, a healthy or normal individual or a comparison. It refers to a value or level of a biomarker in a biological sample that is lower than a value or level range of a biomarker detected in a biological sample obtained from a target individual. It may also be referred to as having a "differential level" or "differential value" or "differently expressed" compared to a "normal" expression level or value of a biomarker, and may be referred to as a quantitative as well as a quantitative difference in expression. Include both sexual differences.

As used herein, the term "biological sample" refers to any sample obtained from a subject in which the expression of the biomarker of the present invention can be detected.

According to a preferred embodiment of the present invention, the biological sample is any one selected from the group consisting of saliva, biopsy, blood, skin tissue, liquid culture, feces and urine, but is not particularly limited thereto, It may be prepared by treatment by a method commonly used in the technical field of the present invention.

Since the method of the present invention is determined to be sensitive using the above-described biomarkers, duplicate descriptions are omitted to avoid excessive complexity in the present specification.

Using the biomarker for predicting susceptibility to OCA, a non-alcoholic fatty liver treatment agent of the present invention, it is possible to reliably determine the susceptibility of each patient before starting treatment, thereby minimizing unnecessary drug administration and increasing treatment efficiency. .

Figure 1a is a schematic diagram showing the entire experimental process for confirming the effect of OCA treatment on mice.
1b to 1d are data comparing average body weight and liver tissue weight of mice administered with Vehicle and OCA for 8 weeks.
Figures 1e to 1g are histopathological comparisons of the decrease in the amount of fat (Steatosis) and inflammation (inflammation) in the liver between an OCA responder group (R) and a non-responder group (NR) before and after OCA treatment.
Figure 1h is data comparing the body weight and liver weight of mice between an OCA responder group (R) and a non-responder group (NR) after OCA treatment.
1i to 1l compare the results of measuring ALT and AST, which are indicators of liver damage, and total cholesterol in serum of mice between an OCA responder group (R) and a non-responder group (NR) after OCA treatment.
Figure 2a is a result of comparing the mRNA expression levels of OCA target genes Nr1h4 (FXR) and Nr0b2 (SHP) in the OCA responder group (R) and non-responder group (NR).
FIG. 2B is data comparing mRNA expression differences of Cytochrom p450 familys (Cyp7a1, Cyp8b1, and Cyp27a1), which are included in the neutral pathway among the two pathways of bile acid synthesis and play a major role in producing bile acids.
FIG. 2c is data comparing the difference in mRNA expression of Cytochrom p450 familys (Cyp7b1, Cyp46a1, and Cyp39a1), which are included in the acidic pathway among the two pathways of bile acid synthesis and play a major role in producing bile acids.
Figure 2d is a comparison of the expression pattern of StarD1, which serves to help cholesterol absorption for the acidic pathway to proceed, in the OCA responder group (R) and the non-responder group (NR).
Figures 2e to 2f compare the expression patterns of Cytochrom p450 familys (Cyp7a1, Cyp8b1, Cyp7b1, and Cyp39a1) involved in the neutral pathway and acidic pathway in the OCA responder group (R) and non-responder group (NR).
Figure 2g divides the expression of each Cytochrom p450 familys gene into above-average expression and below-average expression in mice administered with total OCA, and mice corresponding to the OCA responder group (R) and OCA non-responder group (NR), respectively. It is data showing the ratio of mice.
2h to 2j are the results of examining bile acid composition in the liver.
2k is a result of analyzing the correlation between MCA, Cyp7b1, and Cyp39a1 in order to determine whether a significant increase in acidic pathway metabolites in the OCA responder group (R) was caused by Cyp7b1 and Cyp39a1.
Figure 3a is a result showing the similarity between samples through the PCA plot in the OCA responder group (R) and non-responder group (NR).
Figure 3b is a GSEA analysis result to determine the genetic difference between the OCA responder group (R) and non-responder group (NR).
Figure 3c is data expressing the expression difference of BILE ACID METABOLISM in HALL MARKER as a heat map.
Figure 3d is a result of comparison with mRNA Seq. of Cytochrom p450 familys related to bile acid synthesis.
3E is a comparison result of protein expression of Cyp8b1 and Cyp7b1 in liver tissue biopsied before OCA administration.
3f to 3h show results of comparing the amount of bile acid composition in an OCA responder group (R) and a non-responder group (NR) in liver tissues obtained by tissue biopsy before OCA administration.
3I to 3L show the results of comparing the amount of bile acid composition in the OCA responder group (R) and the non-responder group (NR) in serum before OCA administration.
4a-4b shows the effect of OCA by inhibiting each pathway using siCyp7b1 and siCyp8b1 in LX2 Cell, which is a Human Hepatic Stellate Cell, and then inducing it with TGFβ1 (10 ng/ml) for 24 h, inducing an environment similar to liver fibrosis is the result of comparing
Figures 4c to 4e are the results of comparison of gene expression of TGFβ1, Collagen1, and Fibronectin associated with liver fibrosis under the same conditions as in Figure 4a.
5 is a result of comparing response rates in a responder group (R) and a non-responder group (NR) according to the expression ratio of Cyp7b1 and Cyp8b1.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

Example 1: Comparison of OCA administration effect in non-alcoholic fatty liver disease mouse model

In order to induce a non-alcoholic fatty liver disease mouse model, Western diet (D12079B) was supplied for a total of 24 weeks, and liver tissue biopsy was performed on the 12th week of feeding. Liver tissue at this time was used continuously in the experiment by selecting only mice that were induced to fatty liver through NAS (NAFLD Activity Score), a histopathological observation, and mice that were not induced were sacrificed. (After liver tissue biopsy, NAS and mRNA sequence were performed during the 4-week recovery period.) Fatty liver-induced mice were randomly assigned to the Vehicle group (n=10) and the OCA-administered group (n=20) with 5 mg/kg for 8 weeks. /day was orally administered (Fig. 1a).

After 8 weeks of drug administration, sacrifice was performed and the OCA administered group through the NAS of the liver tissue in each group showed a therapeutic response (OCA R; NAS≤2) and a non-therapeutic response (OCA NR; NAS≥3) were divided and analyzed for comparison.

As a result, the average body weight of mice administered with Vehicle and OCA for 8 weeks was compared over time, and there was no difference in body weight between the two groups (FIG. 1b). However, after 8 weeks of drug administration, it was confirmed that the weight of the liver tissue of mice was significantly reduced in the OCA treatment group compared to the vehicle group (Fig. 1c-d). These results showed a consistent histopathological result of a decrease in the amount of fat in the liver (steatosis) and inflammation (inflammation). When the NR group was divided and compared, the OCA R group was 36.84% and the OCA NR group was 63.16% (Fig. 1e-f).

When the body weight and liver weight of mice in each group were compared, body weight showed no change in each group, but liver weight significantly decreased in the OCA treatment response group (OCAR) (FIG. 1h). Next, ALT and AST, which are indicators of liver damage, were measured in Serum of mice. ALT showed a tendency to decrease in OCAR, but AST did not change. In addition, total cholesterol showed a significant decrease in both the OCA NR and R groups compared to the Vehicle group, but there was no change in triglyceride (Fig. 1i-l).

Example 2: Comparison of activity of genes and proteins involved in the bile acid biosynthetic pathway in mice of a responder group and a non-responder group as a result of OCA administration

It was expected that the cause of the different effects of OCA drugs would depend on the expression of the target gene. When the mRNA expression of the target genes of OCA, Nr1h4 (FXR), Nr2b1 (RXRα), and Nr0b2 (SHP), was compared in each group, there was no significant difference between the two groups. (Fig. 2a). We investigated the difference in mRNA expression of Cyp7a1, another target gene of OCA. Cyp7a1 is known to be an enzyme involved in bile acid synthesis. plays a major role

Although there was no difference between the two groups in the OCA NR and OCA R groups, Cytochrom p450 family members involved in the neutral pathway tended to decrease in the OCA R group (Fig. 2b). (Cyp27a1 is also included in Acidic.)

The composition of bile acid differs depending on the products produced in the two pathways. We compared the gene expression of Cytochrom p450 familys involved in another pathway, the Acidic pathway. As shown in Figure 2c, the gene expression of Cytochrom p450 familys involved in the acidic pathway was increased in the OCA R group.

This acidic pathway proceeds by absorbing cholesterol by StarD1 in the mitochondria, and it was shown that StarD1 significantly increased in gene expression in the OCA R group (FIG. 2d).

It was investigated whether the above results showed the same expression in protein expression. In the two groups, Cyp7a1, Cyp8b1, and Cyp39a1 showed no difference in protein expression, and only Cyp7b1 showed a significant difference. Similar to the Vehicle group, the OCA NR group showed significantly decreased results compared to the OCA R group (Fig. 2e-f).

In addition, the expression of each Cytochrom p450 familys gene in the mice administered with all OCA was divided into above-average expression and below-average expression, respectively, and the ratio of mice corresponding to the OCA R group and OCA NR group was compared. Above-average expression of the Cyp7b1 and Cyp39a1 genes accounted for 71% and 86% of the OCAR mice, whereas among the above-average expression of the OCA NR mice, they accounted for 25% and 17%, respectively (Fig. 2g). These data suggest that mice with increased expression of Cyp7b1 and Cyp39a1 may have an effect on the OCA treatment response group.

Next, bile acid composition in the liver was investigated based on the above results. It was confirmed that CDCA and MCAs, metabolites of the acidic pathway in the liver, were significantly increased in the OCA R group, and total bile acid in the liver was significantly increased in the OCA R group (FIG. 2h-j). To determine whether the significant increase in acidic pathway metabolites in the OCA R group was caused by Cyp7b1 and Cyp39a1, the correlation between MCA, Cyp7b1, and Cyp39a1 was analyzed. Cyp7b1 and a-MCA, b-MCA, and w-MCA showed positive correlations and significant results, but Cyp39a1 did not (FIG. 2k).

Summarizing the above data, it was confirmed that the acidic pathway activity by Cyp7b1 increased in the OCA R group compared to the OCA NR group.

Example 3: Comparison of activity of genes and proteins involved in the bile acid biosynthetic pathway in responder and non-responder mice before OCA administration

The difference between the OCA treatment response group and the non-response group was analyzed to find out through mRNA sequencing using liver tissue biopsies before OCA drug administration (Fig. 3a-d). As a result, first, as a result of showing the similarity between the samples through the PCA plot of each individual, the vehicle showed similarity to OCA NR and OCA R (Fig. 3a). GSEA analysis was conducted to find out the genetic differences between the OCA NR group and the OCA R group. As for the HALL MARKER, BILE ACID METABOLISM and GTPASE REGULATORY ACITIVTY and PROTEIN SERIN THREONINE KINASE ACTIVITY were confirmed in GENE ONTOLOGY. The following is data expressing the difference in expression of BILE ACID METABOLISM in HALL MARKER as a heat map, indirectly confirming that the difference between the OCA R group and the OCA NR group appeared before drug administration (Fig. 3b-c). As a result of comparison with mRNA Seq. of Cytochrom p450 familys related to bile acid synthesis, the expression of Cyp8b1 was lower in the OCA R group than in the OCA NR group (Fig. 3d). We compared the protein expression of Cyp8b1 and Cyp7b1 in liver tissue biopsied before drug administration to investigate whether these results also appeared in proteins. The expression of Cyp8b1 was significantly high in the OCA NR group, but low in the OCA R group, and the expression of Cyp7b1 showed conflicting results (FIG. 3e). As the result above, in the OCA R group, the protein expression of Cyp8b1, the last step of the neutral pathway, decreased, but when the protein expression of Cyp7b1, the last step of the acidic pathway, increased, their metabolite, bile acid composition, also showed the same result. (Fig. 3e-h). We confirmed that Total Bile Acids in the liver were high in the OCA R group, but in Serum, Acidic Bile Acids and Total Bile Acids decreased inversely. In the OCA R group, MCAs and DCA accounted for a high ratio (Fig. 3i-l).

Taken together, it was confirmed that when Cyp7b1 was highly expressed in the liver before OCA administration, the response was excellent when OCA was administered.

Example 4: Comparison of treatment response effects of OCA through inhibition of Cyp7b1 and Cyp8b1 gene expression

Through in vivo results, we inhibited each pathway using siCyp7b1 and siCyp8b1 in LX2 Cell, a human hepatic stellate cell, and then induced it with TGFβ1 (10 ng/ml) for 24 h, resulting in an environment similar to liver fibrosis. induced and compared the effect of OCA.

First, cell proliferation and activation by TGFβ1 were compared through woundhealing assay. Compared to normal, TGFβ1-treated LX2 Cell showed cell proliferation and activation, but OCA 100nM additionally treated group showed suppression. Normal, siCyp7b1 and siCyp8b1 showed similar results, but siC7b1 + TGFb1 and siC8b1 + TGFβ1 showed conflicting results. The group treated with siC7b1 and TGFb1 showed more cell proliferation, but not the group treated with siC8b1 and TGFb1. When 100 nM of OCA was treated here, siC8b1+TGFβ1+OCA inhibited cell proliferation, and siC7b1+TGFβ1+OCA blocked the effect more than TGFβ1+OCA (FIGS. 4a-b).

As a result of comparing gene expression under the same conditions as above, gene expression of TGFβ1, Collagen1, and Fibronectin was significantly increased by TGFβ1, and the above gene expression was decreased in siCyp8b1+OCA, but not in the siCyp7b1+OCA administration group. did In terms of protein expression, FN-1 showed lower expression in siCyp7b1 + TGFβ1 + OCA and siCyp8b1 + TGFβ1 + OCA than in the TGFβ1 + OCA group, but not in Collagen1 (FIG. 4c-e).

Through these results, it was confirmed that the inhibition of Cyp7b1 in the acidic pathway affects the OCA effect.

Example 5: Comparison of response rates in the responder group (R) and the non-responder group (NR) according to the expression ratio of Cyp7b1 and Cyp8b1

Through the above results, it was confirmed that the expression pattern of proteins involved in bile acids metabolism affects the OCA drug response, and in order to obtain more accurate results, we developed a response group (R ), and the non-responder (NR) were analyzed as follows to confirm the correlation.

The ratio of Cyp7b1/Cyp8b1 in each individual of mice administered with total OCA was divided into Response group and Non-Response group and compared. The two groups showed a significant difference. When the ratio was 5 or less, most of the Non-Response group corresponded to this, and when it was 6 or more, the Response group corresponded to this (FIG. 5a).

Finally, most of the mice with a response rate of 36.84% were 71% with above-average Cyp7b1 expression, 57% with below-average Cyp8b1 expression, and 80% when their ratio was 6 or higher (Fig. 5b). ).

Accordingly, it was confirmed that the expression ratio of Cyp7b1 and Cyp8b1 can be used as a marker for predicting whether a patient responds to an OCA drug.

Claims (13)

Cyp7b1 (Cytochrome P450 Family 7 Subfamily B Member 1) gene or a biomarker composition for predicting susceptibility to obeticholic acid (OCA), a treatment for non-alcoholic fatty liver disease, including a protein thereof. According to claim 1, Cyp8b1 (Cytochrome P450 Family 8 Subfamily B Member 1) gene or biomarker composition for predicting sensitivity to non-alcoholic fatty liver disease treatment obeticholic acid (OCA) further comprising a protein thereof. The method according to claim 1, wherein the expression level of the Cyp7b1 gene; Or, if the expression or activity level of its protein is low expression or low activation compared to normal, it is determined that there is resistance to obeticholic acid (OCA),
the expression level of the Cyp7b1 gene; Or, if the expression or activity level of its protein is higher than normal, it is predicted that there is sensitivity to Obeticholic acid (OCA), to non-alcoholic fatty liver disease treatment Obeticholic acid (OCA) A biomarker composition for predicting susceptibility to The non-alcoholic fatty liver according to claim 2, wherein the sensitivity to obeticholic acid (OCA) is predicted according to the ratio of the activity level of the Cyp7b1 gene or its protein and the activity level of the Cyp8b1 gene or its protein. A biomarker composition for predicting susceptibility to disease treatment obeticholic acid (OCA). The non-alcoholic character of claim 4, wherein sensitivity to obeticholic acid (OCA) is predicted when the activity level of the Cyp7b1 gene or its protein is higher than the activity level of the Cyp8b1 gene or its protein. A biomarker composition for predicting sensitivity to obeticholic acid (OCA), a treatment for fatty liver disease. A composition for predicting susceptibility to non-alcoholic fatty liver disease treatment obeticholic acid (OCA), comprising an agent for detecting the biomarker defined in any one of claims 1 to 5. The method of claim 6, wherein the agent for detecting the biomarker is an antisense oligonucleotide, primer pair, or probe that specifically binds to the gene when the biomarker is a gene, and when the biomarker is a protein, the protein A biomarker composition for predicting susceptibility to non-alcoholic fatty liver disease treatment obeticholic acid (OCA), characterized in that it is an antibody, peptide or nucleotide that specifically binds to. A kit for predicting susceptibility to obeticholic acid (OCA), a non-alcoholic fatty liver treatment comprising the composition of claim 6. Method for providing information for predicting sensitivity to obeticholic acid (OCA), a non-alcoholic fatty liver disease treatment comprising the following steps:
(a) preparing a biological sample from the subject;
(b) the expression level of the Cyp7b1 (Cytochrome P450 Family 7 Subfamily B Member 1) gene in the biological sample; Checking the expression or activity level of its protein; and
(c) based on the result of step (b), determining the sensitivity of the subject to obeticholic acid (OCA), a treatment for non-alcoholic fatty liver disease. [Claim 10] The method according to claim 9, wherein step (b) is performed by determining the expression level of the Cyp8b1 gene; Or a method for providing information for predicting sensitivity to non-alcoholic fatty liver disease treatment Obeticholic acid (OCA), further comprising the step of determining the expression or activity level of its protein. 10. The method of claim 9, wherein step (c) is performed by determining the expression level of the Cyp7b1 gene; Or, if the expression or activity level of its protein is low expression or low activation compared to normal, it is determined that there is resistance to obeticholic acid (OCA),
the expression level of the Cyp7b1 gene; Or, if the expression or activity level of its protein is higher than normal, it is predicted that there is sensitivity to Obeticholic acid (OCA), to non-alcoholic fatty liver disease treatment Obeticholic acid (OCA) A method for providing information for predicting susceptibility to 11. The method of claim 10, wherein step (c) predicts the sensitivity to Obeticholic acid (OCA) according to the ratio of the activity level of the Cyp7b1 gene or its protein and the activity level of the Cyp8b1 gene or its protein. A method for providing information for predicting susceptibility to obeticholic acid (OCA), a treatment for non-alcoholic fatty liver disease. 13. The method of claim 12, characterized in that, when the activity level of the Cyp7b1 gene or its protein is higher than the activity level of the Cyp8b1 gene or its protein, it is predicted that there is sensitivity to obeticholic acid (OCA). A method for providing information for predicting susceptibility to obeticholic acid (OCA), a treatment for fatty liver disease.