Trapping assay method and use thereof

Abstract

To achieve a trapping assay method that enables more suitable evaluation of the potential for generating reactive metabolites with respect to candidate compounds including pharmaceuticals.SOLUTION: A trapping assay method according to the present disclosure is a method for trapping assaying reactive metabolites of a test substance by use of CysGlu-Dan, wherein in a test solution comprising the test substance, a metabolic enzyme thereof, and CysGlu-Dan, the concentration of CysGlu-Dan ranges from 0.08 mmol / L to 0.18 mmol / L.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to a trapping assay method and its use. [Background technology]

[0002] Drugs taken into the body are metabolized primarily in the liver, undergoing structural changes that make them more hydrophilic before being excreted from the body. However, metabolic reactions can sometimes produce chemical species that are more reactive than the parent compound. These highly reactive chemical species are called reactive metabolites, and the production of reactive metabolites is considered to be one of the important factors in the development of toxicity. In the drug discovery process, especially in the early discovery stages, trapping assays are often used as screening tests to select compounds with a low potential for reactive metabolite formation. Trapping assays are a technique in which unstable reactive metabolites are reacted with a trapping agent and detected as stable trapping agent adducts.

[0003] CysGlu-Dan is a fluorescent trapping agent used in trapping assays of reactive metabolites. Non-Patent Document 1 describes that 1 mmol / L of CysGlu-Dan can capture and quantify both hard and soft reactive metabolites based on the HSAB (Hard and Soft Acids and Bases) rule. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Chikako Shibazaki, Tomoyuki Ohe, Kyoko Takahashi, Shigeo Nakamura, Tadahiko Mashino, Drug Metabolism and Pharmacokinetics Volume 39, August 2021, 100386. Summary of the Invention [Problem to be solved by the invention]

[0005] However, when the present inventors investigated the concentration dependence of CysGlu-Dan on the quantification of reactive metabolites of various parent compounds (compounds before metabolism are sometimes referred to as "parent compounds" in this specification), they found that, depending on the type of parent compound, the amount of CysGlu-Dan adducts produced by reactive metabolites plateaus or decreases in the presence of high concentrations of CysGlu-Dan. This also revealed that the order of the detected amounts of reactive metabolites can be reversed for multiple parent compounds. Such a reversal of order can make it difficult to appropriately evaluate the reactive metabolite production potential of candidate compounds, such as pharmaceuticals.

[0006] An object of the present invention is to provide a trapping assay method that can more appropriately evaluate the potential for generating reactive metabolites from candidate compounds such as pharmaceuticals. [Means for solving the problem]

[0007] In order to solve the above problems, a trapping assay method according to one aspect of the present invention is a trapping assay method for a reactive metabolite of a test substance using a trapping agent, comprising: The concentration of the trapping agent in the test solution containing the test substance, the metabolic enzyme of the test substance, and the trapping agent is a concentration at which the order of the amounts of trapping agent adducts produced among the multiple test substances is not reversed.

[0008] Furthermore, a trapping assay method according to another embodiment of the present invention is a trapping assay method for a reactive metabolite of a test substance using CysGlu-Dan, in which the concentration of CysGlu-Dan in a test solution containing the test substance, a metabolic enzyme of the test substance, and CysGlu-Dan is 0.08 mmol / L to 0.18 mmol / L. [Effects of the Invention]

[0009] According to an aspect of the present invention, the potential for generating reactive metabolites from a candidate compound such as a pharmaceutical can be more appropriately evaluated. [Brief explanation of the drawings]

[0010] [Figure 1] 1 is a graph showing the relationship between the CysGlu-Dan concentration and the amount of CysGlu-Dan adducts produced as reactive metabolites in the test substances of Examples 1-1 to 1-7. [Figure 2] 1 is a graph showing the relationship between CysGlu-Dan concentration and the residual activity of each CYP molecular species, shown for each substrate of the CYP molecular species. [Figure 3] 2 is a graph showing the relationship between the CysGlu-Dan concentration and the amount of CysGlu-Dan adducts produced as reactive metabolites in the CysGlu-Dan concentration range up to 0.24 mmol / L in the graph of FIG. 1. DETAILED DESCRIPTION OF THE INVENTION

[0011] An embodiment of the present invention will be described below, but the present invention is not limited thereto. In this specification, unless otherwise specified, "A to B" representing a numerical range means "A or more and B or less."

[0012] 1. Trapping Assay Method Non-Patent Document 1 reports that a trapping assay was performed using CysGlu-Dan at a concentration of 1 mmol / L for known reactive metabolites produced in various metabolic reaction systems. However, Non-Patent Document 1 does not discuss in detail what concentration of CysGlu-Dan should be set to in order to evaluate the potential for reactive metabolite production for candidate compounds such as pharmaceuticals.

[0013] When performing reactive metabolite trapping assays using CysGlu-Dan for multiple parent compounds (also referred to herein as "test substances"), it is predicted that varying the concentration of CysGlu-Dan will result in changes in the amount of CysGlu-Dan adducts produced by each reactive metabolite, while maintaining the ranking of CysGlu-Dan adducts among the parent compounds. Therefore, the present inventors have investigated in detail the concentration dependence of the amount of CysGlu-Dan adducts produced by various known parent compounds. As a result, they have independently discovered that, contrary to the above prediction, the amount of CysGlu-Dan adducts produced by reactive metabolites may plateau or decrease in the presence of high concentrations of CysGlu-Dan, resulting in a reversal of the ranking of CysGlu-Dan adducts produced among the parent compounds.

[0014] In trapping assays of candidate compounds such as pharmaceuticals, quantitative values ​​of reactive metabolites are used to evaluate the toxic potential of the candidate compounds. The reversal of the order of CysGlu-Dan adduct formation indicates that the test method is inappropriate for evaluating the toxic potential of such compounds, undermining the significance of trapping assays using CysGlu-Dan. Therefore, determining a CysGlu-Dan concentration that does not result in a reversal of the order of CysGlu-Dan adduct formation is an important challenge in evaluating the reactive metabolite formation potential of candidate compounds such as pharmaceuticals, and is a novel challenge that cannot be achieved based on conventional knowledge.

[0015] Therefore, the present inventors have conducted extensive research into the cause of this reversal in the order of production of CysGlu-Dan adducts, focusing on the possibility that CysGlu-Dan inhibits metabolic enzymes that produce reactive metabolites from parent compounds. As a result, an inhibition test of CYP, a representative metabolic enzyme, has revealed that CYP activity is significantly inhibited by CysGlu-Dan at a concentration of 1 mmol / L. Based on this finding, it is believed that the reversal in the order of production of CysGlu-Dan adducts is due to inhibition of metabolic enzymes by CysGlu-Dan. Note that, although Non-Patent Document 1 has shown that CYP activity is not inhibited at a CysGlu-Dan concentration of 0.1 mmol / L in a CYP inhibition test, the CYP inhibition test was not performed at a CysGlu-Dan concentration of 1 mmol / L, and the presence or absence of CYP activity inhibition was not demonstrated.

[0016] To avoid the inhibition of metabolic enzymes by CysGlu-Dan, it is considered reasonable to reduce the CysGlu-Dan concentration as much as possible. However, if the CysGlu-Dan concentration is reduced too much, it may be difficult to detect the reactive metabolite CysGlu-Dan adducts, depending on the type of parent compound.

[0017] Based on the above findings, the present inventors have found through studies using multiple parent compounds that in order to appropriately evaluate the potential for reactive metabolite formation for candidate compounds such as pharmaceuticals, it is necessary to set a CysGlu-Dan concentration that satisfies the following two requirements: (1) CysGlu-Dan adducts of reactive metabolites can be detected, and (2) the order of the amounts of CysGlu-Dan adducts formed by reactive metabolites is not reversed. Furthermore, they have discovered that a specific range of CysGlu-Dan concentration satisfies the above requirements (1) and (2), leading to the development of a trapping assay method according to one embodiment of the present invention.

[0018] That is, a trapping assay method according to one embodiment of the present invention is a trapping assay method for a reactive metabolite of a test substance using CysGlu-Dan, in which the concentration of CysGlu-Dan in a test solution containing the test substance, a metabolic enzyme of the test substance, and CysGlu-Dan is 0.08 mmol / L to 0.18 mmol / L.

[0019] The CysGlu-Dan concentration range of 0.08 mmol / L to 0.18 mmol / L was obtained by trapping assays using atorvastatin, ketoconazole, tienilic acid, dexamethasone, verapamil, diltiazem, and troglitazone as parent compounds. The determination of the CysGlu-Dan concentration range is described in the Examples.

[0020] The test substance used in the trapping assay method according to one embodiment of the present invention is not particularly limited, but is preferably a drug candidate compound. A drug candidate compound is a compound that is subjected to various tests for the purpose of developing a drug, and is not limited to compounds that form reactive metabolites. Furthermore, the concentration of the test substance in the test solution is not particularly limited, as long as the test substance is soluble in the reaction solution.

[0021] Furthermore, "reactive metabolite" refers to a soft or hard electrophilic substance that is produced by changing the chemical structure of a test substance through metabolism by metabolic enzymes, as described below. "Soft" and "hard" are classifications based on the HSAB rule. Examples of soft electrophilic compounds among reactive metabolites include Michael acceptors such as quinones and epoxides. Examples of hard electrophilic compounds among reactive metabolites include aldehydes and iminiums. CysGlu-Dan can trap both hard and soft reactive metabolites.

[0022] Furthermore, the term "metabolic enzyme" refers to any enzyme derived from human or animal tissue that can metabolize a test substance. The metabolic enzyme used for detecting reactive metabolites is preferably derived from human tissue. Of these, liver tissue is preferred. Examples of metabolic enzymes include cytochrome P450 enzymes (CYP), glucuronosyltransferase, and carboxyesterase. These metabolic enzymes may be used alone or in combination. The concentration of the metabolic enzyme in the test solution is not particularly limited, but is preferably 0.05 mg protein / mL to 2 mg protein / mL, and particularly preferably 1 mg protein / mL.

[0023] The metabolic enzyme may be used in an isolated form or in a form contained in a cell or a cell fraction, and this can be determined appropriately by those skilled in the art.

[0024] Furthermore, the metabolic enzyme contained in cells or cell fractions refers to cells or cell fractions derived from human or animal tissues that contain the metabolic enzyme. Examples of cells or cell fractions that contain the metabolic enzyme include S9 fractions, microsome fractions, and soluble fractions, with cells and microsome fractions being preferred. Liver microsomes are particularly preferred.

[0025] The test solution may contain a coenzyme that assists the function of the metabolic enzyme. By using a metabolic enzyme and a coenzyme in combination, the metabolic efficiency of the test substance by the metabolic enzyme is improved. The coenzyme may be oxidized nicotinamide adenine dinucleotide (NAD + ), oxidized nicotinamide adenine dinucleotide phosphate (NADP + ), and reduced coenzymes such as reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH). Preferably, NADPH or NADP + is used in combination with drug metabolizing enzymes.

[0026] When an oxidized coenzyme is used in combination with a metabolic enzyme, it is preferable to use a substance for regenerating the reduced coenzyme in combination with the metabolic enzyme. For example, NADP + Glucose-6-phosphate (G6P) and glucose-6-phosphate dehydrogenase (G6P-DH) are used in combination.

[0027] In a preferred embodiment, hepatocytes, liver S9 or liver microsomes contain the coenzyme NADPH or NADP. + It is used in combination with liver microsomes and NADPH to detect reactive metabolites. Of these, the combination of liver microsomes and NADPH is more preferred. The concentration of the coenzyme in the test solution is not particularly limited, but is preferably 0.5 mmol / L to 3 mmol / L, and particularly preferably 1.1 mmol / L.

[0028] In the trapping assay method according to one embodiment of the present invention, other features such as the procedure of the trapping assay can be appropriately adopted from conventionally known trapping assays.

[0029] 2. Method for evaluating the reactive metabolite formation potential of a test substance A method for evaluating the reactive metabolite formation potential of a test substance according to one embodiment of the present invention includes, as one step, the trapping assay method according to one embodiment of the present invention described above. In this method, the reactive metabolite formation potential of the test substance is evaluated based on the amount of reactive metabolite detected by the trapping assay method according to one embodiment of the present invention (the amount of CysGlu-Dan adduct formed).

[0030] More specifically, the greater the amount of reactive metabolites detected, the higher the potential of the test substance to produce reactive metabolites is evaluated to be.

[0031] [Embodiment 2] Other embodiments of the present invention are described below.

[0032] The finding that the order of production of CysGlu-Dan adducts as reactive metabolites may be reversed among multiple parent compounds is a unique finding that has not been found in conventional trapping assays using trapping agents other than CysGlu-Dan. The trapping assay method according to another embodiment of the present invention is a method that uses any known trapping agent, not just CysGlu-Dan.

[0033] That is, a trapping assay method according to another embodiment of the present invention is a trapping assay method for the trapping of reactive metabolites of a test substance using a trapping agent, wherein the concentration of the trapping agent in a test solution containing the test substance, a metabolic enzyme of the test substance, and the trapping agent is such that the order of the amounts of trapping agent adducts produced among the multiple test substances is not reversed.

[0034] In another embodiment of the trapping assay method of the present invention, the concentration of the trapping agent is a concentration at which the order of the amounts of trapping agent adducts formed for multiple test substances is not reversed. The method for determining this concentration involves first conducting a trapping assay for multiple test substances, obtaining graph A showing the relationship between the concentration of the trapping agent and the amount of trapping agent adduct formed, and evaluating whether the order of the amounts of trapping agent adducts formed for multiple test substances is reversed. If the order of the amounts of trapping agent adducts formed is reversed, graph B showing the relationship between the residual activity of the metabolic enzyme by the trapping agent obtained by a CYP inhibition test and the concentration of the trapping agent is obtained, and evaluating the concentration dependency of the inhibitory effect of the metabolic enzyme on the trapping agent.

[0035] If the order of the amount of trapping agent adducts produced is reversed based on these evaluation results, a concentration range of the trapping agent that does not reverse the amount of trapping agent adducts produced for multiple test substances is determined from the obtained graph A.

[0036] Examples of trapping agents used in the trapping assay method according to another embodiment of the present invention include glutathione, cysteine, KCN, etc. Furthermore, the multiple test substances used to set the concentration of the trapping agent in the trapping assay method according to another embodiment of the present invention can be appropriately selected depending on the type of reactive metabolite that can be trapped by the trapping agent (whether it is a soft reactive metabolite or a hard reactive metabolite).

[0037] Another embodiment of the method for evaluating the reactive metabolite production potential of a test substance according to the present invention includes the trapping assay method according to the other embodiment of the present invention as one step. This method is similar to the method described in [2. Method for evaluating the reactive metabolite production potential of a test substance], and therefore further description thereof will be omitted.

[0038] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0039] An embodiment of the present invention may include the following features. <1> A trapping assay method for a reactive metabolite of a test substance using a trapping agent, wherein the concentration of the trapping agent in a test solution containing the test substance, a metabolic enzyme of the test substance, and the trapping agent is a concentration that does not reverse the order of the amount of trapping agent adduct produced for multiple test substances. <2> A trapping assay method for a reactive metabolite of a test substance using CysGlu-Dan, wherein the concentration of CysGlu-Dan in a test solution containing the test substance, a metabolic enzyme of the test substance, and CysGlu-Dan is 0.08 mmol / L to 0.18 mmol / L. <3> <1> or <2> A method for evaluating the reactive metabolite production potential of a test substance, comprising the trapping assay method described above as one step. [Example]

[0040] EXAMPLES The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to these examples.

[0041] Example 1: Trapping assay <Compound used> (1) Test substance Atorvastatin calcium trihydrate (Tokyo Chemical Industry Co., Ltd.) Ketoconazole (Sigma-Aldrich) Thienic acid (Sigma-Aldrich) Dexamethasone (Fujifilm Wako Pure Chemical Industries, Ltd.) Verapamil hydrochloride (Sigma-Aldrich) Diltiazem hydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.) Troglitazone (LKT Laboratories) (2) Human liver microsomes (Gentest) (3) Phosphate buffer solution (pH 7.4) (4) CysGlu-Dan (Peptide Institute) (5) β-NADPH (manufactured by Oriental Yeast Co., Ltd.).

[0042] Example 1-1: Trapping assay for atorvastatin As a test substance, atorvastatin calcium trihydrate was dissolved in DMSO (dimethyl sulfoxide) to a concentration of 10 mmol / L to prepare an atorvastatin DMSO solution.

[0043] Next, the atorvastatin DMSO solution, human liver microsomes, phosphate buffer (pH 7.4), CysGlu-Dan, and β-NADPH solution were mixed to prepare a metabolic reaction solution with a final concentration of 120 μL (DMSO concentration at the time of reaction: 1%).

[0044] Atorvastatin calcium trihydrate 100 μmol / L Human liver microsomes 1mg protein / mL Phosphate buffer (pH 7.4) 100mmol / L CysGlu-Dan 1mmol / L β-NADPH 1.1 mmol / L.

[0045] After incubating the metabolic reaction solution at 37°C for 60 minutes, ice-cold methanol (1 mmol / L, containing TCEP) was added in an amount three times the volume of the metabolic reaction solution to terminate the metabolic reaction. The terminated metabolic reaction solution was centrifuged, and the concentration of the reactive metabolite CysGlu-Dan adduct was measured for the supernatant using a UPLC-fluorescence detector according to the analysis described below. TCEP: Tris(2-carboxyethyl)phosphine hydrochloride, a reducing agent.

[0046] (analysis) The concentration of the reactive metabolite CysGlu-Dan adduct was measured using an ultra-high performance liquid chromatography system (Waters) equipped with a fluorescence detector under the following conditions.

[0047] Column: ACQUITY UPLC BEH C18 1.7 μm 2.1 × 100 mm (Waters) Mobile phase A: water / formic acid (1000:2, v / v) Mobile phase B: acetonitrile / formic acid (1000:2, v / v) Measurements were performed using gradient analysis with a total analysis time of 15 minutes. Because fluorescence intensity varies depending on the organic solvent composition, measurements were corrected for the organic solvent composition during elution.

[0048] The concentrations of CysGlu-Dan adducts, reactive metabolites, were measured using the same procedure as above, with the CysGlu-Dan concentrations set at 0.01 mmol / L, 0.03 mmol / L, 0.05 mmol / L, 0.08 mmol / L, 0.1 mmol / L, 0.3 mmol / L, or 0.5 mmol / L.

[0049] Example 1-2: Trapping assay for ketoconazole The concentration of the reactive metabolite CysGlu-Dan adduct was quantified in the same manner as in Example 1-1, except that the test substance was ketoconazole.

[0050] Example 1-3: Trapping assay for tienilic acid The concentration of the reactive metabolite CysGlu-Dan adduct was quantified in the same manner as in Example 1-1, except that the test substance was tienilic acid.

[0051] Example 1-4: Trapping assay for dexamethasone The concentration of the reactive metabolite CysGlu-Dan adduct was quantified in the same manner as in Example 1-1, except that the test substance was dexamethasone.

[0052] Example 1-5: Trapping assay for verapamil hydrochloride The concentration of the reactive metabolite CysGlu-Dan adduct was quantified in the same manner as in Example 1-1, except that the test substance was verapamil hydrochloride.

[0053] Example 1-6: Trapping assay for diltiazem hydrochloride The concentration of the reactive metabolite CysGlu-Dan adduct was quantified in the same manner as in Example 1-1, except that the test substance was diltiazem hydrochloride.

[0054] Example 1-7: Trapping assay for troglitazone The concentration of the CysGlu-Dan adduct, a reactive metabolite, was quantified in the same manner as in Example 1-1, except that the test substance was troglitazone.

[0055] In Examples 1-1 to 1-7, the amounts of CysGlu-Dan adducts, reactive metabolites, produced at each concentration of CysGlu-Dan are shown in Table 1. Table 1 also shows the results when DMSO was used as a negative control for the test compounds.

[0056] [Table 1]

[0057] FIG. 1 is a graph showing the relationship between the CysGlu-Dan concentration and the amount of CysGlu-Dan adduct produced for the test substances of Examples 1-1 to 1-7.

[0058] As shown in Table 1 and Figure 1, the order of the amounts of CysGlu-Dan adducts formed by reactive metabolites of atorvastatin, ketoconazole, tienilic acid, dexamethasone, verapamil, diltiazem, and troglitazone was not maintained at each CysGlu-Dan concentration, but was reversed at certain CysGlu-Dan concentrations. For example, the order of the amounts of CysGlu-Dan adducts formed by reactive metabolites of atorvastatin and dexamethasone was reversed between a CysGlu-Dan concentration of 0.1 mmol / L and a CysGlu-Dan concentration of 0.3 mmol / L.

[0059] Example 2: CYP inhibition experiment <Compound used> (1) Midazolam (Fujifilm Wako Pure Chemical Industries, Ltd.)...CYP3A4 substrate (2) Testosterone (manufactured by the National Institute of Advanced Industrial Science and Technology)...CYP3A4 substrate (3) Amodiaquine dihydrochloride dihydrate (Sigma-Aldrich)...CYP2C8 substrate (4) Bufuralol hydrochloride (Toronto Research Chemicals)...CYP2D6 substrate (5) Human liver microsomes (XenoTech) (6) Phosphate buffer solution (pH 7.4) (7) Magnesium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.) (8) CysGlu-Dan (Peptide Institute) (9) β-NADPH (manufactured by Oriental Yeast Co., Ltd.).

[0060] Example 2-1 A metabolic reaction solution was prepared by mixing midazolam, testosterone, amodiaquine dihydrochloride dihydrate, bufuralol hydrochloride, human liver microsomes, phosphate buffer, magnesium chloride, CysGlu-Dan, and β-NADPH solution to the following final concentrations: Volume of metabolic reaction solution: 100 μL.

[0061] Midazolam 2.5 μmol / L Testosterone 10 μmol / L Amodiaquine dihydrochloride dihydrate 2 μmol / L Bufuralol hydrochloride 5 μmol / L Human liver microsomes 0.2mg protein / mL Phosphate buffer solution (pH 7.4) 98mmol / L Magnesium chloride 3.2 mmol / L CysGlu-Dan 1mmol / L β-NADPH 1.3 mmol / L.

[0062] The metabolic reaction mixture was incubated at 37°C for 10 minutes, and then quenched by adding ice-cold methanol in an amount three times the volume of the metabolic reaction mixture. As a control, a metabolic reaction mixture without CysGlu-Dan was prepared and subjected to the same reaction.

[0063] The metabolic reaction solution after the reaction was stopped was centrifuged, and the metabolites of the substrate in the supernatant were measured by HPLC-MS / MS, and the residual activity of CYP was calculated.

[0064] HPLC-MS / MS analysis conditions: The peak area values ​​of the substrate metabolites were measured using the following system. Liquid chromatography: LC-20A system (Shimadzu Corporation) Mass spectrometer: API4000 (AB Sciex) Column: Inertsil ODS-3, 33 mm L x 2.1 mm ID, 3 μm (GL Sciences) Mobile phase A: 10 mmol / L ammonium acetate aqueous solution (pH 4.9) Mobile phase B: methanol Measurements were performed using a gradient analysis method with a total analysis time of 3.2 minutes.

[0065] Calculation method for the residual activity of CYP: Residual activity rate (%) = (peak area value of substrate metabolite in CysGlu-Dan-added group) / (peak area value of substrate metabolite in control group) × 100.

[0066] The concentrations of CysGlu-Dan were set to 0.008 mmol / L, 0.016 mmol / L, 0.04 mmol / L, 0.08 mmol / L, 0.2 mmol / L, 0.4 mmol / L, or 2 mmol / L, and the substrate metabolites were measured and the residual activity of CYP was calculated using the same procedure as above.

[0067] The residual activity (%) of each CYP molecular species obtained at each concentration of CysGlu-Dan is shown in Table 2. Figure 2 is a graph showing the relationship between the CysGlu-Dan concentration and the residual activity of each CYP molecular species, and is shown for each CYP molecular species substrate.

[0068] [Table 2]

[0069] The results shown in Table 2 and Figure 2 indicate that CysGlu-Dan exhibits an inhibitory effect on metabolic enzymes as its concentration in the test solution increases. In particular, for CYP3A4, it was found that the residual activity of the enzyme decreased to around 20% at a CysGlu-Dan concentration of 1 mmol / L.

[0070] From this, it was considered that the reversal of the order of the production amounts of CysGlu-Dan adducts shown in Example 1 was caused by the inhibition of metabolic enzyme activity by CysGlu-Dan.

[0071] FIG. 3 is a graph showing the relationship between the CysGlu-Dan concentration and the amount of CysGlu-Dan adducts produced as reactive metabolites in the CysGlu-Dan concentration range up to 0.24 mmol / L in the graph of FIG.

[0072] Considering the results of Example 2, it is thought that a trapping assay without reversing the order of production of CysGlu-Dan adducts of reactive metabolites can be achieved by lowering the CysGlu-Dan concentration to avoid metabolic enzyme inhibition by CysGlu-Dan. However, if the CysGlu-Dan concentration is lowered too much, it may be difficult to detect CysGlu-Dan adducts of reactive metabolites, depending on the type of test substance (e.g., tienilic acid shown in Figure 3).

[0073] The graph in Figure 3 shows that by setting the CysGlu-Dan concentration between 0.08 mmol / L and 0.18 mmol / L, it is possible to detect CysGlu-Dan adducts of reactive metabolites for atorvastatin, ketoconazole, tienilic acid, dexamethasone, verapamil, diltiazem, and troglitazone, and that the order of the amounts of CysGlu-Dan adducts produced by reactive metabolites is not reversed. [Industrial Applicability]

[0074] The present invention can be used in the field of drug discovery, which requires a trapping assay.

Claims

1. A trapping assay method for a reactive metabolite of a test substance using a trapping agent, comprising: A trapping assay method in which the concentration of the trapping agent in a test solution containing a test substance, a metabolic enzyme of the test substance, and a trapping agent is a concentration at which the order of the amounts of trapping agent adducts produced for multiple test substances is not reversed.

2. A trapping assay method for reactive metabolites of a test substance using CysGlu-Dan, comprising: A trapping assay method, wherein a test solution containing a test substance, a metabolic enzyme for the test substance, and CysGlu-Dan has a CysGlu-Dan concentration of 0.08 mmol / L to 0.18 mmol / L.

3. A method for evaluating the reactive metabolite production potential of a test substance, comprising the trapping assay method according to claim 1 or 2 as one step.

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