WO2011081214A1 - Method for testing for liver fibrosis by autotaxin assay, and test reagent - Google Patents

Abstract

Provided is a non-invasive and simple method for examining the extent of liver fibrosis with high diagnostic efficiency, and also provided is a test reagent. It is clarified that it is possible to diagnose the extent of liver fibrosis by assaying autotaxin included in a sample, and diagnosis with higher precision is possible by further combination with an existing fibrosis marker.

Description

Test method and test agent for liver fibrosis by autotaxin measurement

The present invention relates to a liver fibrosis test method and a test drug by measuring autotaxin concentration in a human specimen.

Human autotaxin was developed in 1992 by M.M. L. It is a glycoprotein having a molecular weight of about 125 kDa, which was isolated by Strack et al. From A2058 human melanoma cell culture medium as a substance that induces cell motility (J. Biol. Chem. 256, 2524-2529, 1992). Autotaxin produces lysophosphatidic acid (LPA) using lysophosphatidylcholine as a substrate due to its lysophospholipase D activity. Many researchers have shown that LPA is involved in cancer growth and metastasis in vivo (Nat. Rev. Cancer, 3, 582-591, 2003; Int. J. Cancer, 10.109, 833). -838, 2004; Blood, 106, 2138-2146, 2005), the causal relationship between autotaxin, which is its production enzyme, and various diseases has been studied. Recently, a method for quantifying human autotaxin has been established (WO 2008/016186, Clin. Chim. Acta, 388, 51-58, 2008) and is expected as a diagnostic marker for various diseases.
Regarding the relationship between autotaxin and liver disease, it has been reported that serum autotaxin concentration is high in chronic liver disease (Clin. Chim. Acta, 388, 51-58, 2008). There have been reports suggesting that the concentration of autotaxin in human tissues or body fluids may vary depending on various diseases. However, due to the lack of a quantitative method so far, the causal relationship with various diseases has been analyzed. There wasn't. According to the method described in WO2008 / 016186 that we invented, the autotaxin concentration in human specimens such as serum can be quantified simply, in a short time, and with high reliability.
Liver diseases are roughly classified into liver diseases such as non-alcoholic steatohepatitis and the like, and viral hepatitis represented by B and C types. Viral hepatitis is a pathology ranging from acute hepatitis to chronic hepatitis and cirrhosis, but liver fibrosis is involved in the progression of the disease. Fibrosis diagnosis is important in determining treatment policy, predicting the effect, and diagnosing the risk of developing liver cancer. is important. Although a definitive diagnosis of fibrosis is based on a liver biopsy, it is invasive, burdensome to the patient, and difficult to perform frequently. Recently, as a non-invasive examination method, fibroscan (ECHOSENS) or acoustic radiation based on the principle of transient elasticity, which measures the propagation velocity of pulsed vibration waves in tissue with the elasticity (kPa) by ultrasonic image analysis. Ultrasound diagnostic device ACUSON etc. (Mochida Siemens Medical) based on Force Impulse is clinically applied, and it is reported that fibroscan shows good correlation with pathological liver fibrosis (Intervirology, 51 11-16, 2008; Gastroenterology, 128, 343-350, 2005). However, Fibroscan is expensive and can only be seen at specific hospitals and lacks versatility. On the other hand, non-invasive and general-purpose liver fibrosis serum markers such as hyaluronic acid, type IV collagen, type IV collagen 7S, procollagen III peptide, procollagen I peptide, laminin, YKL-40, matrix metal protease 2, TIMP-1, platelet count, indirect serum marker APRI (Aspartate Aminotransferase to Platelet Ratio Index), AAR (Aspartate / alanine aminotransferase ratio), etc. do not show sufficient diagnostic performance compared to fibroscan lo (inter 51) 11-16, 2008).

The present invention uses an invasive and patient-heavy liver biopsy test, which is a problem in diagnosing the degree of liver fibrosis, and an expensive instrument fibroscan installed only in a large facility. The purpose is to solve the problems of blood diagnosis of hyaluronic acid, type IV collagen and the like, which have poor reliability in diagnosis and fibrosis diagnosis, and these diagnostic methods. As a result of intensive studies on the relationship between liver fibrosis and autotaxin concentration in the specimen in order to solve these problems, the present inventors have increased the autotaxin concentration in the specimen in proportion to the degree of liver fibrosis. I found out. Specifically, we found that there was a good correlation between the degree of liver fibrosis by fibroscan and the blood autotaxin concentration, and we found that the blood autotaxin concentration could be a marker reflecting liver fibrosis. . Thus, the present invention provides a test method and a test drug capable of diagnosing the degree of liver fibrosis in a liver disease in a non-invasive, simple and accurate manner by measuring the concentration of autotaxin in human body fluids, particularly serum. To do.

If the measurement reagent described in WO2008 / 016186 invented by the present inventors is used, human autotaxin can be accurately quantified without requiring pretreatment of the specimen. As a result of intensive studies on the relationship between autotaxin concentration and liver fibrosis using this measurement reagent, an increase in autotaxin concentration was observed with liver fibrosis, and it increased in proportion to the degree of fibrosis. I found. Furthermore, it was found that the degree of liver fibrosis can be diagnosed with higher accuracy by combining with existing liver fibrosis markers. This enables highly accurate liver fibrosis diagnosis in a small-scale medical facility in which invasive liver biopsy inspection or expensive fibroscan apparatus installation is difficult.
Specifically, this application includes the following inventions:
(1) A method for examining liver fibrosis, which comprises diagnosing the degree of liver fibrosis by measuring autotaxin concentration in a human specimen.
(2) Diagnosing the degree of liver fibrosis that eliminates the need for male-female differences by measuring the autotaxin concentration in human specimens and using the autotaxin index corrected by the statistical values of each healthy male and female A method for examining liver fibrosis characterized by the following.
(3) The liver fibrosis according to (2), wherein the statistical value described in (2) is any one of an average value, a median value, and a 95th percentile value of autotaxin concentration measurement values Inspection method.
(4) By combining at least one liver fibrosis test and either the autotaxin concentration or the autotaxin index described in (1) to (3), the liver can be diagnosed with higher diagnostic accuracy than the diagnosis by a single test. The method for examining liver fibrosis according to any one of (1) to (3), wherein fibrosis is diagnosed.
(5) The liver fibrosis test described in (4) is hyaluronic acid, type IV collagen, type IV collagen 7S, procollagen III peptide, procollagen I peptide, laminin, YKL-40, matrix metal protease 2, TIMP-1, The method for examining liver fibrosis according to (4), wherein a direct serum marker of any of the platelet counts is used.
(6) The liver fibrosis described in (4), wherein the liver fibrosis test described in (4) uses an indirect serum marker of Aspartate Aminotransferase to Platelet Ratio Index or Aspartate / alanin aminotransferase ratio Inspection method.
(7) The method for inspecting liver fibrosis according to (4), wherein the liver fibrosis test according to (4) is a non-invasive test method selected from among transient elastography and acoustic radiation force impulse.
(8) The specimen described in (1) and (2) is a human blood component such as whole blood, blood cells, serum, plasma, or a human cell or tissue extract (1) to (7) The method for examining liver fibrosis as described.
(9) The method according to (1) to (8), wherein the autotaxin concentration measurement method is an immunochemical method using an antibody.
(10) A liver fibrosis test drug characterized by having the test method described in (1) to (9) as a principle.
(11) The test method according to (1) to (8), wherein the autotaxin concentration measurement method is lysophospholipase D activity.
(12) A liver fibrosis test drug characterized by the principle of the test method according to (11).
(13) A method of determining the degree of liver fibrosis by measuring the autotaxin concentration in a human specimen and comparing it with a standard for the autotaxin concentration of a healthy person.
(14) A method of determining the degree of liver fibrosis by measuring an autotaxin index in a human specimen and comparing it with a standard for an autotaxin index of a healthy person.
(15) A method for examining liver fibrosis by measuring the autotaxin concentration in a human specimen.
(16) A method for determining the degree of liver fibrosis by measuring the autotaxin concentration in a human specimen and comparing it with a standard of autotaxin concentration that indicates each stage of liver fibrosis.
(17) A test kit for liver fibrosis including a reagent for measuring autotaxin concentration and a criterion for autotaxin concentration or index indicating the degree of liver fibrosis.
(18) A liver fibrosis test kit comprising a reagent for measuring lysophospholipase D activity and a criterion for lysophospholipase D activity indicating the degree of liver fibrosis.

According to the present invention, it is possible to diagnose the degree of liver fibrosis by measuring the autotaxin concentration in a human specimen. Autotaxin can be measured by measuring lysophospholipase D activity, which is the enzyme activity of autotaxin, but if it is measured using an immunological quantitative reagent according to the method described in WO2008 / 016186, it will be included in the sample. To provide a test drug that can quantify human autotaxin in a short time without being affected by endogenous measurement interference factors or competing enzymes, and that can be diagnosed easily and at low cost in small-scale medical facilities. Is possible.

FIG. 1 shows the correlation between fibroscan measurements and autotaxin measurements in the serum of male hepatitis C patients.
FIG. 2 shows the correlation between fibroscan measurements and autotaxin measurements in the serum of female hepatitis C patients.
FIG. 3 shows the correlation between fibroscan measurements and autotaxin index across hepatitis C patients. Autotaxin index (ATX index) = autotaxin measured value / 95th percentile upper limit value.
FIG. 4 shows a comparison of the ATX index and the hyaluronic acid concentration (HA) ROC curve when the fibroscan value of 7.1 kPa is taken as the F2 score cutoff value. The solid line indicates the ATX index, the broken line indicates the HA, and the circle indicates the cutoff value of the ATX index and HA.
FIG. 5 shows a comparison of ROC curves composed of the ATX index alone and the product of the ATX index and the HA measurement value when the fibroscan value of 7.1 kPa is used as the F2 score cutoff value. The solid line indicates the ATX index alone, the broken line indicates the product of the ATX index and the HA measurement value, and the circle indicates the cutoff value of the product of the ATX index alone, the ATX index and the HA measurement value.
FIG. 6 shows a comparison of ROC curves composed of the ATX index alone and the product of the ATX index and the APRI measurement value when the fibroscan value 7.1 kPa is taken as the F2 score cutoff value. The solid line indicates the ATX index alone, the broken line indicates the product of the ATX index and the APRI measurement value, and the circle indicates the cutoff value of the product of the ATX index alone, the ATX index and the APRI measurement value.
FIG. 7 shows the correlation between fibroscan measurements and autotaxin measurements in the serum of male hepatitis B patients.
FIG. 8 shows the correlation between fibroscan measurements and autotaxin measurements in the serum of female hepatitis B patients.
FIG. 9 shows the ROC curve of the ATX index when the fibroscan value 12.5 kPa is taken as the F4 score cutoff value.
FIG. 10 shows a distribution diagram of the autotaxin index (ATX index) and the measured hyaluronic acid concentration values of the patient samples in the histological stages F1 to F3 and the patient samples in the F4 group.
FIG. 11 shows ROC curves consisting of autotaxin index (ATX index) and hyaluronic acid concentration measurement values of histological stages F1 to F3 patient specimens and F4 group patient specimens.

An autotaxin quantification reagent and an autotaxin measuring method are described in WO2008 / 016186. The anti-human autotaxin monoclonal antibody used in the quantitative reagent and measurement method can also be obtained by the method described in WO2008 / 016186.
It becomes possible to measure the human autotaxin concentration in the test subject sample using the above quantitative reagent. Here, the correlation coefficient between autotaxin concentration and fibroscan measurement in men is 0.575, and the correlation coefficient between autotaxin concentration and fibroscan measurement in women is 0.674. A good positive correlation has been shown, and the degree of liver fibrosis can be diagnosed by measuring the autotaxin concentration. Although the absolute standard for liver fibrosis diagnosis is liver biopsy, fibrosis diagnosis using fibroscan, a noninvasive diagnostic device, is also used. This is because a very good correlation is obtained. Liver fibrosis can be diagnosed when there is an autotaxin concentration in the subject sample that is significantly higher than the autotaxin concentration of the healthy controls.
In the Man-Whitney U test, there is a significant difference of p <0.001 between the male autotaxin concentration and the female autotaxin concentration. Since it is necessary to consider gender differences when measuring patient samples, the values obtained by dividing patient sample measurements by the 95th percentile upper limit of 0.914 mg / L and 1.323 mg / L for both men and women Autotaxin index (ATX index) = Autotaxin measurement value / 95th percentile upper limit value) When the correlation with the fibroscan measurement value was also investigated for the value corrected for male and female differences, it showed a correlation coefficient of r = 0.644, indicating good correlation Indicated. Therefore, it is possible to diagnose liver fibrosis by measuring the autotaxin index.
The test method for liver fibrosis according to the present invention includes determining the degree of fibrosis. The degree of fibrosis is 5 of F0 (no fibrosis), F1 (portal region fibrosis expansion), F2 (fibrous bridging formation), F3 (fibrous bridging formation with lobular distortion), F4 (cirrhosis). Classified into stages. The absolute standard for diagnosing liver fibrosis is biopsy examination of the liver, but fibrosis diagnosis using fibroscan, which is a noninvasive diagnostic apparatus, is also used. 7.1 kPa is adopted as one of the reference diagnostic criteria for the F2 stage in the fibroscan measurement and 12.5 kPa is adopted as one of the reference criteria for cirrhosis of the F4 stage (Intervirology, 51, 11-16, 2008). Also in the test method of the present invention, the autotaxin concentration and the autotaxin index can be selected as criteria for indicating each step, and based on the criteria, it can be determined to which step the subject belongs. Preferably, it is possible to determine an F2 stage that is a standard of treatment such as interferon and an F4 stage that is a standard of cirrhosis.
The measurement of autotaxin concentration can be replaced with the measurement of lysophospholipase D activity, which is the enzyme activity of autotaxin. In this case, measurement including lysophospholipase D activity other than autotaxin As a true autotaxin-specific quantification method, the value is obtained, the lysophosphatidylcholine substrate used for activity measurement and the choline produced during activity measurement are inherently contained in the sample, etc. Although it is inferior in reliability, it can be used for diagnosis of liver fibrosis. If measurement is performed using an immunological quantitative reagent according to the method described in WO2008 / 016186, it is not affected by endogenous measurement interfering factors or competing enzymes contained in the sample, and in a short time. It is preferable because the human autotaxin concentration can be quantified. A method for measuring lysophospholipase D activity is also described in WO2008 / 016186.
The diagnostic efficiency can be further improved by combining the autotaxin concentration or autotaxin index with other fibrosis markers. Preferably, other fibrosis markers such as hyaluronic acid, type IV collagen, type IV collagen 7S, procollagen III peptide, procollagen I peptide, laminin, YKL-40, matrix metal protease 2, TIMP-1, platelet count, etc. Direct serum markers can be used. Preferably, it can be combined with a hyaluronic acid marker.
In the present specification, the “specimen” means a human blood component such as whole blood, blood cells, serum, plasma, or a human cell or tissue extract, which is collected from a human body.

Examples are shown below, but the present invention is not limited to the examples described in the Examples. In conducting the following experiment, serum samples were collected and measured at the University of Tokyo Hospital with the approval of the Ethics Committee of the University of Tokyo Graduate School of Medicine regarding research ethics. The autotaxin concentration was measured using an automatic immunoassay device AIA series (manufactured by Tosoh Corporation).
Example 1: Measurement of healthy subject specimen Prior to patient specimen measurement, autotaxin concentration of healthy subject serum was measured as a control group. After obtaining informed consent from healthy adult volunteers who have not taken medication 7 days before blood collection, blood is collected from the middle antecubital vein, left at room temperature for 15 minutes, and then centrifuged at 1500 × g for 5 minutes to obtain serum. Acquired and measured. The autotaxin concentration was measured in the serum of 74 healthy men (74 to 23 years old) and 46 women (21 to 60 years old). The measured autotaxin concentration of all 120 people was 0.731 ± 0.176 mg / L (arithmetic mean ± standard deviation), and the reference standard range consisting of the 95th percentile was 0.468 to 1.134 mg / L. . The measured value of male autotaxin concentration was 0.656 ± 0.121 mg / L, the median value was 0.641 mg / L, and the 95th percentile reference standard range was 0.438 to 0.914 mg / L. On the other hand, the measured value of female autotaxin concentration was 0.852 ± 0.184 mg / L, median value 0.820 mg / L, and 95th percentile reference standard range 0.625 to 1.323 mg / L. The autotaxin concentration in female serum was clearly higher than that in males, and Mann-Whitney U test showed a significant difference of P <0.001. Since it is necessary to consider gender differences when measuring patient specimens, the autotaxin index (value obtained by dividing patient specimen measured values by 0.914 mg / L and 1.323 mg / L, which are the upper limits of 95 per-line tiles for each gender) The following evaluation was carried out using a value obtained by correcting the male-female difference in ATX index = autotaxin measurement value / 95th percentile upper limit value).
Example 2: Correlation between autotaxin measurement value and fibroscan measurement value The absolute standard of liver fibrosis diagnosis is biopsy of the liver, and the degree of fibrosis is F0 (no fibrosis), F1 (portal vein fibers) Categorization), F2 (fibrous cross-linking formation), F3 (fibrous cross-linking formation with lobular distortion), and F4 (cirrhosis). Recently, it has become clear that the diagnosis of fibrosis by fibroscan, which is a non-invasive diagnostic device, has a very good correlation with biopsy examination. Therefore, the correlation between the autotaxin concentration and the autotaxin index (ATX index) of each gender and the fibroscan measurement value was verified. Samples of hepatitis C patients were used as samples, and samples with an ALT (alanine aminotransferase) measurement value of 100 IU / L or higher were excluded because there was a report that the fibroscan did not show accurate values. 54 male samples and 70 female samples It examined in. The correlation coefficient between fibroscan and autotaxin concentration in males is r = 0.575 (FIG. 1), the correlation coefficient in females is r = 0.684 (FIG. 2), and the overall correlation is the autotaxin index. The correlation coefficient was r = 0.644 (FIG. 3), and all showed good positive correlation with fibroscan.
Example 3: Diagnosis efficiency of liver fibrosis F2 score or higher by autotaxin index Fibrosis of F2 score or higher is regarded as a standard for treatment of interferon and the like in terms of the degree of liver fibrosis. An important performance index. As one of the reference diagnostic criteria for the F2 stage in fibroscan measurements, 7.1 kPa was classified as a fibrosis group with an F2 score or higher based on 7.1 kPa as a reference (Intervirology, 51, 11-16, 2008) There were 69 positive specimens and 55 negative specimens. In this diagnosis classification, the hyaluronic acid concentration (HA) including the autotaxin index (ATX index), the platelet count, and the diagnosis rate by APRI were evaluated by a statistical method using an ROC curve. As a representative, a comparison of the ATX index and the ROC curve of HA is shown in FIG. In comparison of the AUC values (area under curve) values of the ROC curves, the ATX index was the most excellent at 0.831, and the sensitivity and specificity at that time were 76.81 and 74.55, respectively (Table 1).

Example 4: Diagnosis efficiency of hepatic fibrosis F2 score or more by combination of autotaxin index and other serum markers ATX index and diagnosis efficiency by combination of ATX index and HA, combination of ATX index and APRI using data of Example 3 Compared to a single diagnosis. Since the diagnostic efficiency in the combined method increases with fibrosis, statistical analysis was performed using the product of each marker measurement value. As a result, in any combination, the AUC value was higher than that of the ATX index alone (Table 2, FIGS. 5 and 6).

Further, a significant difference p = 0.05 was shown between the ROC curve by the combination of the ATX index and HA, and the ROC curve by the HA alone, and it was confirmed that the diagnostic efficiency was improved by combining the ATX index.
Example 5: Correlation between autotaxin concentration measurement value and fibroscan measurement value in patients with hepatitis B Correlation between autotaxin concentration measurement value and fibroscan measurement value in hepatitis B virus antigen positive patients as in Example 2. Were examined using 25 male samples and 15 female samples. The correlation coefficient between fibroscan and autotaxin concentration in men is r = 0.660 (FIG. 7), and the correlation coefficient in women is r = 0.522 (FIG. 8). Correlation was shown.
Example 6: Diagnosis efficiency of liver fibrosis F4 score or higher by autotaxin index Fibrosis of F4 score or higher is positioned as liver cirrhosis in the degree of liver fibrosis, and the treatment policy greatly changes, so the diagnosis efficiency of liver cirrhosis of F4 score is diagnosed It becomes an important performance index of the marker. As a reference diagnostic criterion for the F4 stage in fibroscan measurements, 12.5 kPa is classified as a reference (Gastroentrology, 128, 343-350, 2005) and specimens of 12.5 kPa or higher are classified into cirrhosis groups of F4 score or higher There were 80 positive specimens and 44 negative specimens. In this diagnosis classification, the diagnosis rate by the autotaxin index was evaluated by a statistical method using an ROC curve. The AUC value of the ROC curve was 0.858, and the sensitivity at that time was 81.82, the specificity was 73.75, and the effectiveness of liver cirrhosis diagnosis was shown (FIG. 9).
Example 7: Evaluation of diagnostic efficiency using biopsy test specimens Patient serum samples whose histological stage was determined by biopsy examination were used to verify the diagnostic efficiency of cirrhosis in the same manner as in Example 6. Table 3 shows a list of used samples.

Using these specimens, autotaxin concentration and autotaxin index were measured and hyaluronic acid concentration was measured, and the diagnostic efficiency was compared using statistical analysis. FIG. 10 shows a distribution chart comparing the patient specimens of the histological stages F1 to F3 and the patient specimens of the F4 group. In both autotaxin index and hyaluronic acid concentration, the Mann-Whitney U test showed a significant difference of P <0.0009 between the F1 to F3 group patient specimens and the F4 group patient specimens. Was confirmed. Histological stages F1 to F3 group patient specimens and F4 group patient specimens were analyzed by ROC curves, and the curves shown in FIG. 11 were obtained. The autotaxin index and hyaluronic acid concentration AUC / sensitivity were obtained. / Specificity is 0.737 / 71.9% / 71.4% and 0.736 / 56.2% / 82.8%, respectively, confirming that autotaxin measurement is effective in cirrhosis diagnosis did it. Furthermore, the autotaxin index is the cut-off value 2.12 obtained from this ROC analysis, and the hyaluronic acid concentration is the cut-off value 130 μg / L generally used for the diagnosis of cirrhosis. Converted (Table 4). Positive predictive value (number of positive determinations of F4 group patient specimens / positive determination number of all patient specimens × 100), negative predictive value (number of negative determinations of F1 to F3 group patient specimens / negative determination number of all patient specimens × 100) ), The autotaxin index is superior to the hyaluronic acid concentration for both the accuracy ([number of F4 group patient specimens positive + number of F1 to F3 group patient negative judgments] / total number of patient specimens × 100)] It was shown that.

Claims (12)

1. A method for examining liver fibrosis by measuring the autotaxin concentration in human specimens.
2. A test method for liver fibrosis that eliminates the need for male-female differences by measuring the autotaxin concentration in human specimens and using the autotaxin index corrected by the statistical values of healthy male and female individuals.
3. 3. The test for liver fibrosis according to claim 2, wherein the statistical value according to claim 2 is any one of an average value, a median value, and a 95th percentile value of autotaxin concentration measurement values. Method.
4. The combination of at least one liver fibrosis test and either the autotaxin concentration or the autotaxin index according to claims 1 to 3 is more accurate than a single test, according to claims 1 to 3. Examination method of liver fibrosis.
5. The liver fibrosis test according to claim 4, wherein hyaluronic acid, type IV collagen, type IV collagen 7S, procollagen III peptide, procollagen I peptide, laminin, YKL-40, matrix metalloproteinase 2, TIMP-1, platelet count The method for examining liver fibrosis according to claim 4, wherein any one of these direct serum markers is used.
6. The method for inspecting liver fibrosis according to claim 4, wherein the liver fibrosis test according to claim 4 uses an indirect serum marker of Aspartate Aminotransferase to Platelet Ratio Index, Aspartate / alanin aminotransferase ratio. .
7. 5. The method for inspecting liver fibrosis according to claim 4, wherein the liver fibrosis test according to claim 4 is a non-invasive inspection method selected from among transient elastography and acoustic radiation force impulse.
8. The test method according to any one of claims 1 to 7, wherein the specimen according to claims 1 and 2 is a human blood component such as whole blood, blood cells, serum, plasma, or a human cell or tissue extract.
9. 9. The test method according to claim 1, wherein the autotaxin concentration measurement method is an immunochemical method using an antibody.
10. A liver fibrosis test drug characterized by the principle of the test method according to any one of claims 1 to 9.
11. 9. The test method according to claim 1, wherein the autotaxin concentration measurement method measures lysophospholipase D activity.
12. A liver fibrosis test drug characterized by the principle of the test method according to claim 11.

Images