JP2012002610A - Biomarker for inspecting liver damage and method for predicting liver damage by using biomarker - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a biomarker capable of inspecting drug-induced liver damage, especially glutathione excess use type liver damage, and to provide a method for predicting and diagnosing the drug-induced liver damage, especially the glutathione excess use type liver damage, by detecting the biomarker or determining the quantity of the biomarker and measuring an appearance form of the biomarker.SOLUTION: The biomarker for inspecting liver damage is composed of cholic acid, glycocholic acid and taurocholic acid.

Description

  The present invention relates to a biomarker for liver injury test. More specifically, the present invention relates to a biomarker for testing drug-induced liver injury. The present invention uses a change in the balance of the bile acid biosynthesis pathway as an indicator to detect or quantify a biomarker capable of examining drug-induced liver damage, particularly glutathione overuse (depletion type) liver damage, and the biomarker. In addition, the present invention relates to a method for predicting drug-induced liver injury by measuring its expression pattern.

Conventionally, in a toxicity evaluation test of a drug candidate compound, the compound was administered to an experimental animal such as a rat, and the histopathological change was examined by an optical microscope or an electron microscope. However, histopathological changes often do not become apparent unless they are administered for a long period of time, and further, there are drawbacks such as the time and labor required for preparation and detection of tissue specimens.
Moreover, in the toxicity test in a clinical trial and the diagnosis of the side effect in a medication patient, the application of the above-mentioned method requiring the collection of a biopsy specimen is very limited because of the large surgical invasion.
Accordingly, there is a need for the development of an evaluation system that can efficiently and non-invasively predict or diagnose drug-induced toxicity.

  Metabolomics, which comprehensively analyzes peripheral body fluids (urine, plasma, etc.) or intermediate / final metabolites in organs and cells, is a method for capturing changes in biological responses that come to the next stage of transcriptomics / proteomics. It is being used in various fields of medicine and biology. In the field of toxicology, this technology has begun to be used for elucidation of the mechanism of toxicity and prediction of toxicity. Together with toxicogenomics and toxicoproteomics, conventional toxicological endpoints (symptoms, clinical tests, histopathology) It is expected to be applied to drug safety assessment and clinical diagnosis as a technique that provides suggestions for molecular toxicological end points in place of clinical testing.

  On the other hand, there is glutathione conjugation as a typical route responsible for detoxification in vivo. Glutathione is thought to be excreted as mercapturic acid when a harmful substance in the body binds to glutathione and glutamate and glycine are cut off. Glutathione is also known as an antioxidant component, scavenging radicals, regulating cell functions by redox, and SH donors of various enzymes.

  For example, acetaminophen, which is an antipyretic analgesic along with aminopyrine and aspirin, is excreted in urine as unchanged substance, and 90-95% undergoes glucuronidation or sulfate conjugation in the liver in the urine. It is excreted. The rest is oxidized with cytochrome P-450 to N-acetyl parabenzoquinonimine (NAPQI) which causes hepatocyte necrosis. This NAPQI is normally conjugated and detoxified by glutathione (GSH) in hepatocytes, and further metabolized to be excreted in the urine as the final products mercapto acid and cysteine. However, when a large amount of acetaminophen is present in the body, glucuronidation or sulfate conjugation is saturated, metabolism via cytochrome P-450 proceeds, and production of NAPQI increases. As NAPQI increases, glutathione required for detoxification is depleted and cannot be completely detoxified, resulting in liver damage.

  That is, in a situation where the intracellular glutathione concentration is low, there is a high possibility of drug toxicity, and therefore a biomarker for glutathione depletion is required.

  When glutathione in the liver is consumed by binding to poisons or foreign substances, γ-glutamylcysteine synthase (γ-GC) is activated and glutathione is synthesized. At the same time, ophthalmic acid is biosynthesized in the liver using 2-aminobutyric acid as a substrate by γ-GC and glutathione synthetase and excreted in the blood. When liver glutathione is depleted, blood ophthalmic acid rapidly increases, and it has been reported in mice that ophthalmic acid in blood can be a biomarker indicating depletion of liver glutathione (Patent Document 1, Non-Patent Document 1). ).

  However, ophthalmic acid is a biomarker linked to glutathione synthesis in the liver, and does not directly indicate that glutathione in the liver is depleted. The increase in the blood concentration of phthalate is ahead of the increase in AST (aspartate aminotransferase) and ALT (alanine aminotransferase), which are used in general biochemical tests as markers of liver damage. It is not a definitive diagnosis because it is observed. Accordingly, there remains a need for new glutathione overutilized biomarkers that can better reflect glutathione depletion in the liver.

  In addition, AST and ALT, which are markers of liver damage used in general biochemical tests, increase the blood concentration due to the destruction of hepatocytes regardless of the cause, so drug-induced liver damage, especially glutathione It was insufficient as a biomarker capable of specifically diagnosing overuse liver damage.

JP 2007-192746 A

T. Soga et al., J. Biol. Chem. 2006 281, 16768-16776

  The present invention relates to a biomarker capable of examining drug-induced liver damage, particularly glutathione overuse liver damage, and drug-induced liver damage, particularly glutathione, by detecting or quantifying the biomarker and measuring its expression pattern. It is an object of the present invention to provide a method for predicting and diagnosing overuse liver damage.

In recent years, cross-omics analysis has attracted attention as a technique that enables more effective drug design and compound safety studies. Cross-omics analysis integrates various omic data such as metabolomics to analyze metabolites, proteomics to analyze enzymes and regulatory proteins, genomics to analyze regulatory genes such as transcription and expression, and transcriptomics To deal with.
In order to solve the above-mentioned problems, the present inventors have performed a cross-omics analysis on the liver and plasma of an acetaminophen liver injury model (ie, glutathione overuse liver injury model) using rats generally used in toxicity tests ( Genomics, metabolomics).
As a result, the following characteristics were observed in the biosynthetic pathway of bile acids in the glutathione overuse liver injury model.
(1) The blood concentration of primary bile acid increases.
(2) The composition ratio of primary bile acids changes (those with taurocholic acid> glycocholic acid are reversed with taurocholic acid <glycocholic acid).
Furthermore, by performing microarray analysis and pathological examination using RNA extracted from the liver, it was confirmed that such changes in the primary bile acid biosynthetic pathway can be an indicator of glutathione overuse liver injury.
As a result of further studies based on these findings, the present inventors have completed the present invention. In metabolomics, analysis is usually performed by paying attention to components in which changes are observed, but the present inventors also pay attention to components in which changes are not observed and combine various metabolic pathways related to the components in a complex manner. By studying, I succeeded in finding a new indicator.

That is, the present invention is as follows.
[1] A biomarker for liver injury testing, comprising primary bile acid.
[2] The biomarker according to the above [1], wherein the liver disorder is glutathione overutilization type liver disorder.
[3] The biomarker according to [1] or [2] above, wherein the primary bile acids are cholic acid, glycocholic acid and taurocholic acid.
[4] The biomarker according to [1] or [2] above, wherein the primary bile acids are chenodeoxycholic acid, glycochenodeoxycholic acid and taurochenodeoxycholic acid.
[5] A method for predicting liver damage by a compound,
(1) a step of detecting cholic acid, glycocholic acid and taurocholic acid in a sample collected from a mammal to which the compound has been administered and comparing the amounts thereof with a control;
(2) a step of measuring the constituent ratio of glycocholic acid / taurocholic acid in the sample, and (3) a step of predicting the liver injury-inducing property of the compound using the results of (1) and (2) as an index,
Including methods.
[6] A method for predicting liver damage by a compound,
(1) a step of detecting chenodeoxycholic acid, glycochenodeoxycholic acid and taurochenodeoxycholic acid in a sample collected from a mammal to which the compound has been administered, and comparing the amount with a control;
(2) a step of measuring the composition ratio of glycochenodeoxycholic acid / taurochenodeoxycholic acid in the sample, and (3) a step of predicting the liver injury-inducing property of the compound using the results of (1) and (2) as an index. ,
Including methods.
[7] In step (1), the total amount of cholic acid, glycocholic acid and taurocholic acid in the sample is significantly larger than that of the control, and in step (2), the constituent ratio of glycocholic acid / taurocholic acid is 1 The method according to [5] above, wherein it is determined that the compound induces liver damage when the ratio is larger than.
[8] In step (1), the total amount of chenodeoxycholic acid, glycochenodeoxycholic acid and taurochenodeoxycholic acid in the sample is significantly larger than the control, and in step (2), the composition ratio of glycochenodeoxycholic acid / taurochenodeoxycholic acid The method according to [6] above, wherein the compound is judged to induce liver damage when is greater than 1.
[9] The method according to any one of [5] to [8] above, wherein the liver disorder is glutathione overutilization type liver disorder.
[10] The method according to any one of [5] to [9] above, wherein the sample is serum or plasma.
[11] A method for diagnosing glutathione depletion,
(1) detecting cholic acid, glycocholic acid and taurocholic acid in a sample collected from a mammal to be diagnosed, and comparing the amount with a sample (control) collected from a healthy mammal;
(2) a step of measuring a constituent ratio of glycocholic acid / taurocholic acid in the sample, and (3) a step of diagnosing whether glutathione is depleted using the results of (1) and (2) as an index,
Including methods.
[12] A method of diagnosing glutathione depletion,
(1) detecting chenodeoxycholic acid, glycochenodeoxycholic acid and taurochenodeoxycholic acid in a sample collected from a mammal to be diagnosed, and comparing the amount with a sample (control) collected from a healthy mammal;
(2) a step of measuring the composition ratio of glycochenodeoxycholic acid / taurochenodeoxycholic acid in the sample, and (3) diagnosis of whether the compound is glutathione-depleted using the results of (1) and (2) as an index The process of
Including methods.
[13] In step (1), the total amount of cholic acid, glycocholic acid and taurocholic acid in the sample is significantly larger than that of the control, and in step (2), the constituent ratio of glycocholic acid / taurocholic acid is 1. The method according to [11] above, wherein a diagnosis of glutathione-depleted state is made when greater than.
[14] In step (1), the total amount of chenodeoxycholic acid, glycochenodeoxycholic acid and taurochenodeoxycholic acid in the sample is significantly larger than the control, and in step (2), the composition ratio of glycochenodeoxycholic acid / taurochenodeoxycholic acid The method according to [12] above, wherein when the value is larger than 1, diagnosis of glutathione depletion is made.
[15] The method according to any one of [11] to [14] above, wherein the sample is serum or plasma.

  The biomarker of the present invention is useful for evaluating the presence or absence of liver damage, particularly glutathione overuse liver damage. Since glutathione plays an important role in glutathione conjugation, which is a kind of detoxification action in the liver, and depletion of the substance leads to side effects of the drug, the biomarker of the present invention is useful as a bimarker of glutathione depletion. It can also be suitably used for screening for compounds that do not induce glutathione overuse liver damage. Furthermore, the biomarkers of the present invention are useful for predicting liver damage induced by compounds.

It is a schematic diagram which shows the biosynthetic pathway of a bile acid in a mammal (KEGG map). It is a figure which shows the result of having investigated the fluctuation | variation of various bimarkers at the time of acetaminophen hepatotoxicity induction. AST and ALT were used as biochemical markers. As indices of histopathological changes, EC (acidophilic change of cells), Nec (necrosis), Inf (inflammation of inflammatory cells), and Swl (cell swelling) were examined. Cholic acid, taurocholic acid and glycocholic acid are the biomarkers of the present invention. It is a graph which shows the result of having measured the quantity of the biomarker of this invention in a liver and plasma at the time of acetaminophen hepatotoxicity induction. It is a schematic diagram which shows the biosynthetic pathway of the primary bile acid in a liver. It is a schematic diagram showing the biosynthetic pathway of primary bile acids in the liver when acetaminophen hepatotoxicity is induced. It is the figure which showed typically the criterion of the glutathione overutilization type liver disorder. It is a schematic diagram which shows the procedure of sample (plasma sample) preparation from the rat to which acetaminophen was orally administered. It is a schematic diagram which shows the procedure of sample (liver sample) preparation from the rat to which acetaminophen is orally administered.

The present invention provides a biomarker for liver injury testing, comprising primary bile acid.
Bile acids include primary bile acids such as cholic acid and chenodeoxycholic acid, which are bile acids synthesized in the liver, and secondary bile acids (deoxycholic acid and lithocholic acid) produced from primary bile acids by intestinal bacteria in the small intestine. Acid). When cholic acid derived from cholesterol is conjugated with taurine (amide bond), taurocholic acid is generated, and when conjugated with glycine (peptide bond), glycocholic acid is generated. This conjugation reaction involves bile acid CoA: amino acid-N-acyltransferase (EC 2.3.3.15).

Bile acids normally enter the bile as taurocholic acid or glycocholic acid.
FIG. 1 shows a biosynthetic pathway (KEGG map) of bile acids in mammals. In the present invention, the primary bile acids include free forms of cholic acid and chenodeoxycholic acid, taurine conjugated types, and glycine conjugated types, with cholic acid, glycocholic acid and taurocholic acid being preferred. Also preferred are combinations of chenodeoxycholic acid, glycochenodeoxycholic acid and taurochenodeoxycholic acid.

  The liver disorder to be tested with the biomarker of the present invention is a liver disorder in which a significant increase in the blood concentration of at least a marker of liver disorder (eg, AST or ALT) used in normal biochemical tests is observed. Yes, preferably a liver disorder in which an increase in the blood concentration of primary bile acid is observed in addition to a significant increase in the blood concentration of AST or ALT, more preferably in addition to their pathological condition, Liver disorder with fluctuations. Here, “variation in the composition ratio of bile acids” means that in the normal case, taurocholic acid (TCA)> glycocholic acid (GCA) is reversed to GCA> TCA. That is, it is a liver disorder having a pathological condition in which the composition ratio of GCA / TCA is larger than 1. Specifically, glutathione overuse liver damage.

Variations in the composition ratio of bile acids can also be observed in the combination of chenodeoxycholic acid, glycochenodeoxycholic acid and taurochenodeoxycholic acid. That is, in the normal case, taurochenodeoxycholic acid> glycochenodeoxycholic acid, but in the case of glutathione overutilization type liver injury, it reverses to glycochenodeoxycholic acid> taurochenodeoxycholic acid.
Hereinafter, for the sake of convenience, the present invention will be described in the case where the primary bile acid is a combination of cholic acid, glycocholic acid and taurocholic acid, but the biobacteria of the present invention are similarly applied to a combination of chenodeoxycholic acid, glycochenodeoxycholic acid and taurochenodeoxycholic acid. It can be used as a marker and as a tool for the prediction method and diagnosis method of the present invention.
In the present specification, the “component ratio” corresponds to the “concentration ratio” when each component is represented by the concentration in the sample (specimen).

  Typical liver damage caused by glutathione overuse is caused by drugs such as acetaminophen, rifampicin, phenytoin, bromobenzene, sulfasalazine, methyltestosterone, flutamide, methimazole, dilutiazem, dantrolene, danazol, and phenacetin. Is mentioned. Furthermore, glutathione depletion has been reported in γ-glutamylcysteine synthetase deficiency (OMIM 230450) and glutathione synthetase deficiency (OMIM 266130) (Blood 95: 2193-96 (2000); J Pediatrics 139: 79-84 ( 2001) etc.). Decreased glutathione has also been reported in Parkinson's disease and AIDS (Progress in Neuro-Psychopharmacology and Biological Psychiatry 20: 1159-70 (1996); Lancet 334, Issue 8675: 1294-1298 (1989)).

  The biomarker of the present invention can be used for the examination / diagnosis of liver disorders with such a clear mechanism of action, and also predicts the presence or absence of liver disorders, particularly whether glutathione overuse liver damage -It can be suitably used for diagnosis.

  The biomarker of the present invention is derived from a mammal. Examples of mammals include, for example, laboratory animals such as rodents and rabbits such as mice, rats, hamsters, and guinea pigs, domestic animals such as pigs, cows, goats, horses, and sheep, pets such as dogs and cats, humans, monkeys, Primates such as orangutans and chimpanzees. The mammal is preferably a primate, more preferably a human. In addition, when the biomarker of the present invention is used for prediction of compound toxicity and screening of compounds as described later, laboratory animals such as mice and rats are preferably used.

The history of identification of primary bile acids, particularly cholic acid, glycocholic acid and taurocholic acid, as biomarkers for liver injury testing of the present invention is shown below.
The present inventors used liver and plasma of rats orally administered with acetaminophen (APAP), which has been reported to induce hepatotoxicity in the process of searching for a safety biomarker by toxicogenomics. Metabolomics was performed. APAP was orally administered to rats, and liver and plasma were collected over time. From the liver and plasma samples, metabolomics of 514 kinds of metabolic standards using a capillary electrophoresis mass spectrometer (CE-TOFMS) was extracted from the liver, and RNA was extracted from the liver separately for microarray analysis. As a result, it was confirmed that fluctuations in the amount of bile acids and their composition ratio occurred in relation to depletion of glutathione in the liver. Based on the above, primary bile acid is used as a biomarker for testing liver damage, especially glutathione overuse liver damage, or as an index marker for predicting whether the administered compound will induce glutathione overuse liver damage. obtain.

  The examination of liver injury using the biomarker of the present invention is based on detecting or quantifying primary bile acid contained in a biological sample collected from a subject. In this test, the biological specimen includes peripheral body fluids (urine, plasma, serum, etc.) collected from the subject, preferably the plasma or serum.

The method for detecting or quantifying the biomarker of the present invention in a biological sample is not particularly limited as long as it is a method capable of detecting or quantifying the biomarker. Here, quantification means quantification of the amount (concentration in the sample) of primary bile acids, particularly cholic acid, glycocholic acid and taurocholic acid, which are biomarkers. Detection and quantification of each component may be performed by any method, and each component may be detected simultaneously or separately. Examples of methods for simultaneously detecting each component include nuclear magnetic resonance (eg, ¹ H NMR, ¹³ C NMR), gas chromatography (GC) method, gas chromatography-mass spectrometry (GC-MS) method, high performance liquid Chromatography (HPLC) method, HPLC-NMR method, liquid chromatography-mass spectrometry (LC-MS) method, LC-MS-MS method, thin layer chromatography-mass spectrometry (TLC-MS) method, capillary electrophoresis mass An analyzer (CE-TOFMS), a method using an immunoassay (radioimmunoassay, etc.) and the like can be mentioned, but not limited thereto. The measurement conditions such as various parameters in each detection method can be easily selected by those skilled in the art depending on the type of the measurement substance.

The present invention provides a method for predicting liver damage caused by a compound using the biomarker. Specifically, the prediction method includes the following steps.
Step 1: detecting cholic acid, glycocholic acid and taurocholic acid in a sample collected from a mammal to which the compound has been administered, and comparing the amounts with a control;
Step 2: a step of measuring the constituent ratio of glycocholic acid / taurocholic acid in the sample, and step 3: a step of predicting the liver injury-inducing property of the compound using the results of Step 1 and Step 2 as an index

The mammal to which the prediction method of the present invention is applied is not particularly limited as long as the compound has been administered in advance, and examples thereof include rodents such as mice, rats, hamsters, guinea pigs, and rabbits. Laboratory animals, domestic animals such as pigs, cows, goats, horses and sheep, pets such as dogs and cats, and primates such as humans, monkeys, orangutans and chimpanzees. The sex, age, weight, etc. of the animal are not particularly limited and vary depending on the animal species. For example, in the case of humans, normally, healthy adult males are preferably selected in the phase I study from the viewpoint of maternal protection (female・ This is not the case for drugs specific to children and anticancer agents). In the case of rats, those of about 2 to about 24 months of age and weight of about 100 to about 700 g are preferably used, but are not limited thereto.
When the mammal is non-human, it is preferred to use a genetically and microbiologically controlled animal population. For example, genetically, it is preferable to use inbred and closed colony animals. In the case of rats, for example, inbred rats such as Sprague-Dawley (SD), Wistar, LEW, etc., and in the case of mice, Examples include, but are not limited to, inbred mice such as BALB / c, C57BL / 6, C3H / He, DBA / 2, SJL, and CBA, and closed colony mice such as DDY and ICR. Microbiologically, it may be a conventional animal, but from the viewpoint of eliminating the influence of infectious diseases, it is more preferable to use SPF (specific pathogen free) or gnotobiotic grade.

Examples of the compound to be administered to mammals include pharmaceutical or veterinary drug candidate compounds (compounds in clinical trials when the test animal is a human).
The method of administering the compound to the mammal is not particularly limited. For example, the test compound is orally or parenterally in the form of a solid, semi-solid, liquid, aerosol, etc. (eg, intravenous, intramuscular, intraperitoneal, artery) (Internal, subcutaneous, intradermal, intratracheal, etc.). The dose of the test compound varies depending on the type of compound, animal species, body weight, dosage form, administration period, etc. For example, when administered for a short period of about 3 days, the cells of the target organ survive as long as the animal can survive. The amount necessary to be able to be exposed to the highest concentration of test compound possible for a period of time or more. In clinical trials, various dosages are selected within a range set based on data obtained in preclinical trials. Administration can be performed once to several times. The time from administration to sampling varies depending on the animal species, the dose of test compound, pharmacokinetics, etc. For example, in the case of rats, when a high dose is administered for a short period, it is preferably about 1 to about 7 days from the initial administration. Is about 3 to about 5 days. When a low dose is administered for a long period, about 1 month or more, preferably about 2 to about 6 months from the initial administration can be mentioned.

  Feeding / water supply during the administration period, breeding method such as light / dark cycle are not particularly limited. For example, in the case of rats and mice, a commercially available solid or powdered feed and fresh tap water or well water are freely ingested, and the light period is 12 hours. / A method of rearing in a 12-hour dark cycle is mentioned. If necessary, the food can be fasted and / or water-free for a certain period of time.

  The sample collected from the mammal used in the prediction method of the present invention is not particularly limited as long as it is a biological sample from which primary bile acids, particularly cholic acid, glycocholic acid and taurocholic acid can be detected. It is preferable to use peripheral body fluids (urine, plasma, serum), particularly plasma or serum, since the invasion to the test animal is small and the change in the composition ratio of bile acids linked to glutathione depletion is remarkably observed.

  As a method for collecting peripheral body fluid, for example, in the case of urine, a normal urinalysis method can be used for humans. The urine can be forcibly collected or spontaneously excreted urine within a certain time (for example, about 1 to about 24 hours, preferably about 3 to about 12 hours) can be collected in a urine collection container using a metabolic cage. . The latter is preferable in that a special technique is not required and data variation is small. When storing urine, the urine collection container is preferably ice-cooled in order to prevent changes in urinary metabolites, and a small amount of toluene, thymol, concentrated hydrochloric acid or the like may be dropped as a preservative. Furthermore, a small amount of liquid paraffin can be added to the urine collection container in order to prevent urine evaporation. The collected urine is subjected to measurement after purification of the supernatant by centrifugation or the like, if necessary. However, if it takes time until measurement, it may be stored frozen and thawed before use.

  In the case of plasma, venous blood is collected from the back of the elbow or back of the hand by a normal blood collection method in the case of humans, and in the case of non-human mammals, blood that has flowed out by injuring the tail vein, tail artery, orbital venous plexus Can be prepared by collecting blood cells using a capillary tube, etc., and adding and removing anticoagulants such as heparin, EDTA, sodium citrate as necessary, and separating and removing blood cells by centrifugation or filtration through a plasma separation membrane. . In the case of serum, the collected blood can be prepared by allowing the collected blood to stand for a certain period of time to form a clot and then collecting the supernatant by centrifugation or the like. If it takes time to measure both plasma and serum, it can be frozen and thawed before use.

In the prediction method of the present invention, cholic acid (CA), glycocholic acid (GCA) and taurocholic acid (TCA) in the specimen obtained as described above are detected and quantified.
In mammals, cholic acid is converted to TCA and GCA, respectively, by taurine conjugation or glycine conjugation by the action of bile acid CoA: amino acid-N-acyltransferase (EC 2.3.3.1.65) (FIG. 4). ). In the normal case, the amount relationship of TCA and GCA in the blood is GCA <TCA, whereas in the case of glutathione depletion, the total amount of bile acids (CA, GCA and TCA) is In addition to a significant increase, the inventors have found that the quantitative relationship is reversed as GCA> TCA, that is, the GCA / TCA composition ratio is greater than 1 in a glutathione-depleted state. Here, “significantly” means that the difference is statistically significant when compared to the control.
Therefore, the amount of CA, GCA and TCA in the specimen is measured, and the composition ratio between GCA and TCA is measured and compared with that in the case of no compound administration (control). As a result, (1) CA, GCA And the total amount of TCA (CA + GCA + TCA) and (2) the GCA / TCA composition ratio is greater than 1, it can be predicted that the compound is glutathione overutilized hepatopathy-inducing.
CA, GCA and TCA may be detected by any measurement method, and each component may be detected simultaneously or separately. Specifically, the same method as described above is used as a method for detecting or quantifying the biomarker of the present invention.

  The accuracy of the prediction method of the present invention can be evaluated by observing histopathological changes. Furthermore, microarray analysis using RNA extracted from the liver can be simultaneously performed and confirmed based on the results.

  According to the prediction method of the present invention, even in a relatively short-term measurement of about one day, the composition ratio of GCA / TCA is larger than 1, and it is predicted that glutathione overuse type liver injury is induced. can do. As described above, the prediction method of the present invention enables determination with high accuracy of positive / negative in a shorter period of time, and therefore can improve the screening efficiency of the developed compound in the initial stage of drug development, In the clinical trial stage, the risk of developing hepatotoxicity can be predicted before specific symptoms are developed, and the risk of the subject can be reduced.

The present invention provides a method for diagnosing whether or not a glutathione-depleted state using the biomarker. The diagnostic method specifically includes the following steps.
(1) detecting cholic acid, glycocholic acid and taurocholic acid in a sample collected from a mammal to be diagnosed, and comparing the amount with a sample (control) collected from a healthy mammal;
(2) a step of measuring the constituent ratio of glycocholic acid / taurocholic acid in the sample, and (3) a step of diagnosing whether or not glutathione is depleted using the results of (1) and (2) as an index ,
Including methods.
Here, the glutathione depletion state is observed as described above, such as a marked decrease in the glutathione concentration in the liver caused by various drugs such as acetaminophen, or γ-glutamylcysteine synthetase deficiency or glutathione synthetase deficiency. Refers to a marked decrease in glutathione concentration in the liver.
Specifically, the diagnosis subject is a patient who has been administered a drug suspected of depleting glutathione or causing glutathione depletion as a side effect. “Healthy” means a case where it is not recognized that liver damage is caused by evaluation with other biomarkers or histopathological evaluation.
Each step is performed in the same manner as each step in the method for predicting liver damage of the present invention described above.
Finally, in the diagnostic method, in step 1, the total amount of primary bile acids (cholic acid (CA), glycocholic acid (GCA) and taurocholic acid (TCA)) in the sample is significantly higher than that of the control. In many cases, the process 2 includes the step of determining that the glutathione overuse liver disorder has occurred when the composition ratio of GCA / TCA is larger than 1. Although the total amount of primary bile acids (CA + GCA + TCA) in the sample is significantly higher than that of the control, in Step 2, when the GCA / TCA composition ratio is 1 or less, the liver damage is induced by liver damage. However, it is predicted that it is not a glutathione overuse liver disorder.
The criteria for determining glutathione overuse liver damage are schematically shown in FIG.

The present invention also provides a kit that can be suitably used in the prediction method and diagnosis method of the present invention. The kit of the present invention comprises a reagent for measuring primary bile acids, particularly cholic acid, glycocholic acid and taurocholic acid. The measuring reagent is not particularly limited as long as it enables quantitative analysis. Specifically, a reagent capable of performing the above-described method as a method for detecting or quantifying the biomarker of the present invention is included.
EXAMPLES The present invention will be described more specifically with reference to the following examples. However, these are merely examples and do not limit the scope of the present invention.

(1) Materials and methods <Metabolomics of rats administered with acetaminophen (APAP)>
The measurement was performed using liver samples and plasma samples of APAP-administered rats.
A total of 514 metabolic standards (cationic 293 types, anionic 191 types) under the analysis conditions of three types of capillary electrophoresis-mass spectrometer (CE-MS) for cation measurement, anion measurement, and nucleotide measurement , 30 kinds of nucleotides) were measured, and metabolites in liver and plasma after 3, 6, 9, 24 hours after administration of APAP oral administration (1000 mg / kg) rats and control rats were measured using this standard substance. Identified and quantified. A two-sided test (T test) was performed on the measurement results of APAP oral administration rats and control rats at each time.

1. Sample Preparation from Rats Administered APAP Orally or Control Rats Male rats (Crl: CD (SD) IGS; 6 weeks old) were orally administered APAP (1000 mg / kg body weight) or physiological saline as a control once. Breeding with free water and free feeding. Liver and plasma were collected from rats 3, 6, 9, 24 hours after oral APAP administration. Five rats per group were used at each point.

2. Sample pretreatment and measurement (plasma)
50 μL of plasma was added to 450 μL of methanol solution prepared so that methionine sulfone, MES and CSA (10-camphorsulfonic acid) were each 20 μM, and stirred. Further, 500 μL of chloroform and 200 μL of milli-Q water were added and stirred, followed by centrifugation at 4 ° C. and 4,600 g for 5 minutes. As much as possible from the upper water-methanol layer was taken in an ultrafiltration filter (fractionated molecular weight 5,000 Da) and centrifuged at 9,100 g for 2 hours at 4 ° C. A sample obtained by centrifugal concentration of 200 μL of the filtrate was used as a sample. Before measurement, 3-aminopyrrolidine and trimesate were dissolved in 25 μL of an aqueous solution containing 200 μM of each and measured under the CE-TOFMS measurement conditions shown in Tables 1 and 2. The procedure for sample preparation from blood is shown in FIG.

3. Sample pretreatment and measurement (liver)
Liver tissue (about 30-60 mg) was immersed in 625 μL of a methanol solution prepared to 20 μM each of methionine sulfone, MES and CSA (10-camphorsulfonic acid), and zirconia beads (5 mm × 2, 3 mm × 4) were added. Used to disrupt the tissue. The amount of supernatant obtained from the water-methanol layer was taken into an ultrafiltration filter (fractionated molecular weight: 5,000 Da) and centrifuged at 9,100 g for 3 hours at 4 ° C. A sample obtained by centrifugal concentration of 300 μL of the filtrate was used as a sample. Before the measurement, 50 μL of an aqueous solution containing 3-aminopyrrolidine and trimesate each 200 μM was dissolved and measured under the CE-TOFMS measurement conditions shown in Tables 1 and 2. The procedure for sample preparation from tissue is shown in FIG.

4). Quantification of sample The metabolite standard solution (STD) was prepared to the concentrations shown in Table 3, and the sample was measured after measuring the STD corresponding to the beginning of each measurement condition.

  The concentration of each component in plasma was calculated according to the internal standard calibration method using the following formula, assuming that the internal standard substance (IS) concentration in the suspension of methanol and plasma was 18 μM.

  The concentration of each component in the liver was calculated according to the internal standard calibration method using the following formula, assuming that the IS concentration in methanol after crushing the liver was 20 μM. The number of moles in 625 μL of methanol was determined from the determined concentration in methanol, and divided by the weight (g) of each sample to determine the number of moles per gram of liver.

<Biochemical examination of rats treated with acetaminophen (APAP)>
For APAP oral administration rats or control rats used in the above metabolomics, blood concentrations of AST and ALT, which are generally used as markers of liver damage, were measured 3, 6, 9 and 24 hours after APAP administration. .
AST and ALT were measured using methods commonly practiced in the art. That is, AST and ALT were measured by JSCC standard method.
<Histopathological findings of rats treated with acetaminophen (APAP)>
APAP oral administration rats or control rats used in the above metabolomics were evaluated histopathologically 3, 6, 9 and 24 hours after APAP administration.
The histopathological evaluation was performed by observing the presence and extent of eosinophilic changes, necrosis, inflammatory cell infiltration and cell swelling of hepatocytes. By fixing the liver tissue collected at each time point with 10 vol% neutral buffered formalin, embedding in paraffin according to a conventional method, preparing a sliced specimen, staining with hematoxylin and eosin, and observing the liver tissue and / or hepatocytes under a microscope evaluated.
<Analysis at the gene level>
After normalizing the numerical value of gene expression measured using GeneChip of Affymetrix Co. using the MAS5.0 algorithm, logarithm conversion with Log2 is performed on the control data processed in the same manner. The expression level of each molecule at the level was confirmed.
For example, the expression level of bile acid CoA: amino acid-N-acyltransferase (EC 2.3.1.65), which will be described later, is determined by using Affymetrix's GeneChip (Rat Genome 230 2.0 Array) and rat liver and plasma fractions. Using, the nucleic acid molecules contained in each fraction were hybridized with the nucleic acid array on GeneChip, labeled with one or more label components, and measured by detecting the hybridized polynucleotide complex.

(2) Results 24 hours after the oral administration of APAP, AST and ALT increased 1.5 to 2.7 times, and at that time, hepatic acidification, necrosis and hepatocellular enlargement centered on lobule In addition, hepatocyte infiltration was observed. That is, it was confirmed that hepatotoxicity occurred 24 hours after oral administration of APAP (FIG. 2).
Metabolomics allowed identification and quantification of 226 metabolites from liver and 147 metabolites from plasma. 109 significant changes were detected in the liver compared to the control and 60 significant variations in the plasma. At 24 hours after administration, an increase in ophthalmic acid and its precursors was observed simultaneously with a decrease in reduced glutathione in the liver.
Furthermore, a significant variation was observed in the primary bile acid concentration in plasma (FIG. 2).
As is clear from FIG. 2, significant increases in plasma concentrations of cholic acid and glycocholic acid were observed 24 hours after oral APAP administration (at which time hepatotoxicity occurred). On the other hand, taurocholic acid did not change significantly in its plasma concentration even 24 hours after oral administration of APAP, but cells were dead due to hepatotoxicity (obtained from an increase in AST and ALT, histopathological evaluation) In view of this, it was considered that the production amount of taurocholic acid actually increased about twice as much as that observed per hepatocyte.
From the above findings, it was shown that, together with cholic acid and glycocholic acid, taurocholic acid can also function as a biomarker of liver damage, particularly glutathione overuse liver damage.

  In order to clarify the function of the primary bile acid as a biomarker, the total plasma concentration of cholic acid, glycocholic acid and taurocholic acid (hereinafter also referred to as “total amount of primary bile acids” for convenience) is shown in a bar graph. A combination of plasma concentrations of glycocholic acid and taurocholic acid in a line graph was combined. The results are shown in FIG. In all the samples collected from rats that were orally administered APAP, the total amount of primary bile acids in plasma increased compared to the control, and the composition ratio of primary bile acids was further changed. Administration of APAP resulted in a plasma concentration of glycocholic acid> taurocholic acid (= glycocholic acid / taurocholic acid component ratio greater than 1). On the other hand, in the rats not treated with APAP, except for the control 4 in which the total amount of primary bile acids in the plasma was extremely small, all showed the opposite results to the APAP-treated rats in which glycocholic acid <taurocholic acid.

As is clear from FIGS. 3 and 4, cysteine acts on both glutathione production pathway and cholesterol metabolism pathway in liver metabolism. By administering APAP, the contribution of cysteine to the glutathione production pathway preferentially progresses, and the amount of taurine used in the cholesterol metabolic pathway decreases accordingly (the amount of taurine is measured by metabolomics as with primary bile acids). . On the other hand, the amount of glycine does not decrease (the amount of glycine is also measured by metabolomics). Further, bile acid CoA, which is an enzyme used for conjugating taurine to cholic acid or glycine in the cholesterol metabolic pathway: The expression level of amino acid-N-acyltransferase (EC 2.3.3.15) is almost constant by APAP administration.
Therefore, the depletion state of glutathione by APAP administration leads to a decrease in taurine, and the amount relationship between taurocholic acid and glycocholic acid in plasma is reversed accordingly.
Based on these findings, a criterion for determination of glutathione overuse liver damage was obtained (FIG. 6).

  As a result of analyzing the metabolomics data, it was found that the four genes shown in FIG. 4 are related, and discriminant analysis was performed using gene expression information obtained from toxicogenomics. First, using literature information, etc., a learning set was made by classifying into compounds that cause glutathione overutilization depletion (12) and compounds that do not cause glutathione depletion (46). Here, they are called a positive compound and a negative compound, respectively. Next, using linear discrimination, it was confirmed by discriminant analysis that positive compounds and negative compounds can be classified by the expression variation of 4 genes, and the compounds including the learning set were predicted. The following 4 genes were used.

Gclc: Gamma-glutamylcysteine synthetase (EC 6.3.2.2)
Gss: Glutathione synthetase (EC 6.3.2.3)
Cyp7a1: Cytochrome P450, family 7, subfamily A (EC 2.3.1.65)
Baat: Bile acid-CoA: amino acid N-acyltransferase (EC 1.14.13.17)

An example of discriminating analysis results with a high discrimination rate is shown below.
If the variable name is 1370688_at_Gclc_24_3hr,
1370688_at is the Affymetrix probe ID,
Gclc is an abbreviation for genes,
24_3hr indicates the difference between the expression value for 24 hours and the expression value for 3 hours.
The variables of other discriminant functions are displayed according to the same rule. Table 4 shows the types of variables and their coefficients, and Table 5 shows the discrimination results.

By projecting gene expression values other than positive negative compounds to the discriminant space, ranking was performed from positive compounds to negative compounds or vice versa. Positive compounds were ranked in the upper center (Table 6: Table 6-1 and Table 6-2).
These results show the validity of selecting four genes, Gclc, Gss, Cyp7a1, and Baat, as genes involved in glutathione depletion, and the interrelationship of their expression fluctuations causes depletion due to excessive use of glutathione. It has been shown to be reasonable for screening compounds.
Table 6 shows the results of excerpting the score order compounds obtained from the results of the linear discriminant analysis. The learning set used in the linear discrimination was expressed as Posi and Nega (all displayed). Compounds other than the learning set were described as unknown (10 for each of the upper and lower ranks).

  INDUSTRIAL APPLICABILITY The present invention is useful for evaluating the presence or absence of liver damage, particularly glutathione overuse liver damage, and thus predicts liver damage induced liver damage induced by glutathione overuse and screens for safer drugs. It becomes possible to do.

Claims (15)

  A biomarker for liver injury testing, consisting of primary bile acids.   The biomarker according to claim 1, wherein the liver disorder is glutathione overutilization type liver disorder.   The biomarker according to claim 1 or 2, wherein the primary bile acids are cholic acid, glycocholic acid and taurocholic acid.   The biomarker according to claim 1 or 2, wherein the primary bile acids are chenodeoxycholic acid, glycochenodeoxycholic acid and taurochenodeoxycholic acid. A method for predicting liver damage caused by a compound,
(1) a step of detecting cholic acid, glycocholic acid and taurocholic acid in a sample collected from a mammal to which the compound has been administered and comparing the amounts thereof with a control;
(2) a step of measuring the constituent ratio of glycocholic acid / taurocholic acid in the sample, and (3) a step of predicting the liver injury-inducing property of the compound using the results of (1) and (2) as an index,
Including methods. A method for predicting liver damage caused by a compound,
(1) a step of detecting chenodeoxycholic acid, glycochenodeoxycholic acid and taurochenodeoxycholic acid in a sample collected from a mammal to which the compound has been administered, and comparing the amount with a control;
(2) a step of measuring the composition ratio of glycochenodeoxycholic acid / taurochenodeoxycholic acid in the sample, and (3) a step of predicting the liver injury-inducing property of the compound using the results of (1) and (2) as an index. ,
Including methods.   In step (1), the total amount of cholic acid, glycocholic acid and taurocholic acid in the sample is significantly larger than the control, and in step (2), the constituent ratio of glycocholic acid / taurocholic acid is greater than 1. 6. The method according to claim 5, characterized in that it is determined that the compound induces liver damage.   In step (1), the total amount of chenodeoxycholic acid, glycochenodeoxycholic acid and taurochenodeoxycholic acid in the sample is significantly larger than the control, and in step (2), the constituent ratio of glycochenodeoxycholic acid / taurochenodeoxycholic acid is 1 The method according to claim 6, wherein it is determined that the compound induces liver damage when the compound is larger.   The method according to any one of claims 5 to 8, wherein the liver disorder is glutathione overutilization type liver disorder.   The method according to any one of claims 5 to 9, wherein the sample is serum or plasma. A method for diagnosing glutathione depletion, comprising:
(1) detecting cholic acid, glycocholic acid and taurocholic acid in a sample collected from a mammal to be diagnosed, and comparing the amount with a sample (control) collected from a healthy mammal;
(2) a step of measuring a constituent ratio of glycocholic acid / taurocholic acid in the sample, and (3) a step of diagnosing whether glutathione is depleted using the results of (1) and (2) as an index,
Including methods. A method for diagnosing glutathione depletion, comprising:
(1) detecting chenodeoxycholic acid, glycochenodeoxycholic acid and taurochenodeoxycholic acid in a sample collected from a mammal to be diagnosed, and comparing the amount with a sample (control) collected from a healthy mammal;
(2) a step of measuring the composition ratio of glycochenodeoxycholic acid / taurochenodeoxycholic acid in the sample, and (3) diagnosis of whether the compound is glutathione-depleted using the results of (1) and (2) as an index The process of
Including methods.   In step (1), the total amount of cholic acid, glycocholic acid and taurocholic acid in the sample is significantly larger than the control, and in step (2), the constituent ratio of glycocholic acid / taurocholic acid is greater than 1. The method according to claim 11, characterized in that it is diagnosed as being glutathione depleted.   In step (1), the total amount of chenodeoxycholic acid, glycochenodeoxycholic acid and taurochenodeoxycholic acid in the sample is significantly larger than the control, and in step (2), the constituent ratio of glycochenodeoxycholic acid / taurochenodeoxycholic acid is 1 The method according to claim 12, wherein the method is diagnosed as being depleted of glutathione.   The method according to any one of claims 11 to 14, wherein the sample is serum or plasma.