Luminescence system and method for quantifying cytochrome P450

Abstract

The purpose of the present invention is to provide a cytochrome P450 light emission system which can easily be made to emit light without the need for excitation light; this purpose is met by a light emitting system characterized by containing cytochrome P450 and a heterocyclic compound represented by general formula (1) or a salt thereof [in the formula, R1 is a hydrogen or a C1-4 alkyl group, R2 is NR4 2 or OH; the R4s are independently a hydrogen or a C1-6 alkyl group, and two R4s of the NR4 2s may optionally bond together and form a ring; A is represented by general formula (2) or (3), the R3s are independently CR5 or N; R5s are independently a hydrogen, a C1-8 alkyl group or a C2-8 alkenyl group, and n is an integer 0-3].

Description

[Technical Field] 【0001】 This invention relates to a light emission system and a method for quantifying cytochrome P450. [Background technology] 【0002】 Cytochrome P450 enzymes are metabolic enzymes present in almost all living organisms, from bacteria to plants and mammals, and are involved in drug and toxin metabolism, hormone biosynthesis, fatty acid metabolism, and secondary metabolism in plants. In drug discovery, elucidating the process by which drugs are metabolized by cytochrome P450 is extremely important for considering side effects and drug interactions (Non-Patent Literature 1). 【0003】 Conventionally, fluorescence assays and luminescence assays have been known as methods for detecting the activity of cytochrome P450. In fluorescence assays, substrates that produce fluorescent products after metabolism by cytochrome P450 are used. Here, test compounds that modulate cytochrome P450 activity are identified by their effect on the accumulation of the fluorescent product. On the other hand, the luminescence assay utilizes the luminescence reaction between firefly luciferase and its substrate, firefly luciferin (Non-Patent Literature 2). Specifically, a derivative of firefly luciferin that is not a substrate of firefly luciferase is prepared in advance, and in the first reaction, cytochrome P450 converts the derivative of firefly luciferin into firefly luciferin. Then, in the second reaction, the generated firefly luciferin reacts with firefly luciferase to produce light, and the amount of light emitted is measured to quantify the cytochrome P450. [Prior art documents] [Non-patent literature] 【0004】 [Non-Patent Document 1] "Drug Interactions Mediated by Cytochrome P450," Pharmacia, 31(9), 992-996, (1995) [Non-Patent Document 2] "Screening of Cytochrome P450 Activity Using Luminescence Assay Method", Promega Corporation, November 19, 2006 【Summary of the Invention】 【Problems to be Solved by the Invention】 【0005】 However, the excitation light required for the fluorescence assay generates a background signal, which limits the assay sensitivity. On the other hand, since the luminescence assay does not require excitation light, the background is kept low and the assay sensitivity is high. However, gene introduction for the expression of firefly luciferase into the target is necessary. Therefore, in in vitro (in vitro experiments), it is necessary to add and act firefly luciferase, and in the case of in vivo (in vivo experiments), it is necessary to prepare a genetically modified organism into which the firefly luciferase gene has been introduced in advance, and it is not possible to emit light simply. 【0006】 Therefore, an object of the present invention is to solve the above problems of the prior art and provide a luminescence system for cytochrome P450 that does not require excitation light and can emit light simply. Another object of the present invention is to provide a method for quantifying cytochrome P450 simply and with high sensitivity using the above luminescence system. 【Means for Solving the Problems】 【0007】 As a result of intensive studies to solve the above problems, the present inventors have found that cytochrome P450 reacts with a compound having a specific structure to emit light, and by quantifying the light emission, cytochrome P450 can be quantified, and have completed the present invention. That is, the gist configuration of the present invention for solving the above problems is as follows. 【0008】 The luminescence system of the present invention is represented by the following general formula (1): 【Chemical formula】 [In the formula, R , 5 , , 【0011】 , , , , , 1 , 【0009】 , 5 , , , , 【0010】 , , , , , is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, R 2 is NR 4 2 or OH, where R 4 is each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and NR 4 2's two R 4 may be bonded to each other to form a ring, A is the following general formula (2) or (3): 【Chemical formula】 represented by, R 3 is each independently CR 5 or N, where R 5 is each independently a hydrogen atom, an alkyl group having 1 to 8 carbon atoms or an alkenyl group having 2 to 8 carbon atoms, n is an integer of 0 to 3], and is characterized by comprising a heterocyclic compound represented by the formula or a salt thereof and cytochrome P450. Such a luminescence system of the present invention does not require excitation light and can easily obtain luminescence derived from cytochrome P450. 【0009】 In a preferred example of the luminescence system of the present invention, the cytochrome P450 is present in a living tissue. In this case, luminescence derived from cytochrome P450 in the living tissue can be obtained. 【0010】 In another preferred example of the luminescence system of the present invention, the cytochrome P450 is present in mammalian liver tissue. In this case, luminescence derived from cytochrome P450 in mammalian liver tissue can be obtained. 【0011】 The method for quantifying cytochrome P450 of the present invention comprises reacting cytochrome P450 with the following general formula (1): 【Chemical formula】​​​​​​​​2 , NR 4 2 or OH, where R 4 Each is independently either hydrogen or an alkyl group having 1 to 6 carbon atoms, and NR 4 Two R's 4 They may be joined to each other to form a ring. A is given by the following general formula (2) or (3): [ka] It is represented as, R 3 Each of them operates independently in CR 5 Or N, where R 5 These are, independently, hydrogen, an alkyl group having 1 to 8 carbon atoms, or an alkenyl group having 2 to 8 carbon atoms. A heterocyclic compound represented by [n is an integer between 0 and 3] or a salt thereof is reacted, and the amount of light emitted is measured. The method is characterized by measuring the amount of cytochrome P450 from the amount of light emitted. The present invention provides a simple and highly sensitive method for quantifying cytochrome P450. [Effects of the Invention] 【0012】 According to the present invention, a cytochrome P450 light emission system can be provided that does not require excitation light and can be easily emitted. Furthermore, the present invention provides a simple and highly sensitive method for quantifying cytochrome P450. [Brief explanation of the drawing] 【0013】 [Figure 1] This shows the results of measuring the amount of luminescence in a system containing arthropod extracts and the compound of structural formula (1-1) or firefly luciferin. [Figure 2] This shows the results of measuring the amount of luminescence in mammalian liver culture cells and a system containing the compound of structural formula (1-1) or firefly luciferin. [Figure 3]This shows the results of measuring the decrease in luminescence in a system containing a pill bug (male) extract, a cytochrome P450 inhibitor, and the compound shown in structural formula (1-1). [Figure 4] This shows the results of measuring the decrease in luminescence in a system containing a pill bug (female) extract, a cytochrome P450 inhibitor, and the compound shown in structural formula (1-1). [Figure 5] This shows the results of measuring the decrease in luminescence in a system containing a male black blowfly extract, a cytochrome P450 inhibitor, and the compound shown in structural formula (1-1). [Figure 6] This shows the results of measuring the decrease in luminescence in a system containing a female black blowfly extract, a cytochrome P450 inhibitor, and the compound shown in structural formula (1-1). [Figure 7] This shows the results of measuring the decrease in luminescence in a system containing mouse liver extract, a cytochrome P450 inhibitor, and the compound shown in structural formula (1-1). [Figure 8] The results show the reduction in luminescence when mice were administered a cytochrome P450 inhibitor and the compound shown in structural formula (1-2) (in vivo). [Figure 9] This shows the results of measuring the amount of luminescence in a system containing CYP1A2 and a heterocyclic compound represented by general formula (1) or a salt thereof. [Figure 10] This shows the results of measuring the amount of luminescence in a system containing CYP2A6 and a heterocyclic compound represented by general formula (1). [Figure 11] This shows the results of measuring the amount of luminescence in a system containing CYP2B6 and a heterocyclic compound represented by general formula (1) or a salt thereof. [Figure 12] This shows the results of measuring the amount of luminescence in a system containing CYP2C9 and a heterocyclic compound represented by general formula (1) or a salt thereof. [Figure 13] This shows the results of measuring the amount of luminescence in a system containing CYP2D6 and a heterocyclic compound represented by general formula (1) or a salt thereof. [Figure 14] This shows the results of measuring the amount of luminescence in a system containing CYP2E1 and a heterocyclic compound represented by general formula (1) or a salt thereof. [Figure 15]This shows the results of measuring the amount of light emitted in a system containing CYP3A4 and a heterocyclic compound represented by general formula (1) or a salt thereof. [Figure 16] The results shown are in vivo, after administering the compound with structural formula (1-2) (D-form) or the compound with structural formula (1-8) (L-form) to mice and measuring the amount of luminescence. [Figure 17] These are bioluminescence images (in vivo) taken after administering the compound with structural formula (1-2) to black mallow fly larvae. [Figure 18] The results (in vivo) show the amount of luminescence measured after administering the compound shown in structural formula (1-2) to a mouse model of chronic liver disease induced by carbon tetrachloride and a control group of mice. [Figure 19] The results (in vivo) show the luminescence levels measured after administering the compound shown in structural formula (1-2) to non-alcoholic fatty liver disease model mice and control mice. [Modes for carrying out the invention] 【0014】 The luminescence system and cytochrome P450 quantitative method of the present invention will be described in detail below, based on embodiments thereof. 【0015】 <Light-emitting system> The light-emitting system of the present invention is defined by the following general formula (1): [ka] [In the formula, R 1 is hydrogen or an alkyl group having 1 to 4 carbon atoms. R 2 , NR 4 2 or OH, where R 4 Each is independently either hydrogen or an alkyl group having 1 to 6 carbon atoms, and NR 4 Two R's 4 They may be joined to each other to form a ring. A is given by the following general formula (2) or (3): [ka] It is represented as, R 3 Each of them operates independently in CR 5 Or N, where R 5 These are, independently, hydrogen, an alkyl group having 1 to 8 carbon atoms, or an alkenyl group having 2 to 8 carbon atoms. The present invention is characterized by comprising a heterocyclic compound or a salt thereof represented by [n is an integer between 0 and 3], and cytochrome P450. 【0016】 In the light emission system of the present invention, a heterocyclic compound represented by general formula (1) or a salt thereof reacts with cytochrome P450 to produce light. Unlike fluorescence assays, the light emission system of the present invention does not require excitation light. Furthermore, unlike luminescence systems using firefly luciferin, the luminescence system of the present invention does not require firefly luciferase. Therefore, even in vivo (in living organisms), it is not necessary to create genetically modified organisms into which the firefly luciferase gene has been introduced in advance. Therefore, the light-emitting system of the present invention does not require excitation light and can emit light easily. 【0017】 The luminescence system of the present invention provides a method for directly visualizing the activity of cytochrome P450. When cytochrome P450 is treated with the heterocyclic compound represented by the general formula (1) or a salt thereof that reacts with the cytochrome P450, it emits light according to the activity level of the cytochrome P450. Unlike the luminescence system of fireflies, this luminescence does not require luciferase, and therefore does not require the introduction of a luciferase gene into the object being measured. Furthermore, by combining a specific cytochrome P450 with the heterocyclic compound represented by the general formula (1) or a salt thereof that interacts with it, it becomes possible to sense cytochrome P450 that is widely present in living organisms. 【0018】 (Heterocyclic compounds or salts thereof) In the above general formula (1), R 1R is hydrogen or an alkyl group having 1 to 4 carbon atoms. Examples of alkyl groups having 1 to 4 carbon atoms include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, etc. From the viewpoint of luminescence efficiency, 1 Hydrogen is preferred as the primary gas. Note -COOR 1 Regarding the chiral carbon of the thiazoline ring to which the compound is bonded, the stereochemistry can be either R or S. 【0019】 In the above general formula (1), R 2 , NR 4 2 or OH, where R 4 Each is independently either hydrogen or an alkyl group having 1 to 6 carbon atoms, and NR 4 Two R's 4 These may be joined to each other to form a ring. 4 Regarding this, examples of alkyl groups having 1 to 6 carbon atoms include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, etc. Also, NR 4 Two R's 4 The following formula is used to form the cyclic structure that is formed when these elements bond together with N: [ka] Examples include the 1-azacyclopropyl group (three-membered ring), 1-azacyclobutyl group (four-membered ring), 1-azacyclopentyl group (five-membered ring), 1-azacyclohexyl group (six-membered ring), and 1-azacycloheptyl group (seven-membered ring), represented by R. 4 A methyl group is preferred as the component. R 2 From the perspective of luminous efficiency, NR 4 2 is preferred, and N(CH3)2 is particularly preferred. 【0020】 In the above general formula (1), A is represented by the above general formula (2) or (3). Note that, from the viewpoint of luminous efficiency, A is represented by the above general formula (2) or (3), and R 3 CR 5It is preferable that it is represented by the above general formula (2). 【0021】 In the above general formulas (2) and (3), R 3 Each of them operates independently in CR 5 Or N, where R 5 Each of these is independently a hydrogen atom, a C1-C8 alkyl group, or a C2-C8 alkenyl group. 5 Regarding this, examples of alkyl groups having 1 to 8 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, and octyl groups, while examples of alkenyl groups having 2 to 8 carbon atoms include vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, pentenyl, hexenyl, heptenyl, and octenyl groups. From the standpoint of ease of synthesis, R 3 CR 5 It is preferable that this is the case. Also, from the viewpoint of solubility in water and buffer solutions with a pH near neutral, R 3 It is preferable that one or more of these values ​​are N. 【0022】 In the general formula (1) above, n represents the number of repeating vinylene units (-CH=CH-) and is an integer from 0 to 3. As the number of n increases, the emission wavelength becomes longer. Therefore, from the viewpoint of visualizing deep tissues in living organisms, n is preferably 2 or 3, and from the viewpoint of ease of synthesis, n is preferably 2. 【0023】 The heterocyclic compounds represented by the above general formula (1) or salts thereof can be synthesized, for example, according to the methods disclosed in Japanese Patent Publication No. 5464311, Japanese Patent Publication No. 6011974, Japanese Patent Publication No. 6353751, and International Publication No. 2013 / 027770, or commercially available products can be used. 【0024】 The heterocyclic compound represented by the above general formula (1) can also be in the form of a salt. The salt of the heterocyclic compound represented by general formula (1) can also react with cytochrome P450 to emit light. Here, the salt of the heterocyclic compound represented by the general formula (1) above may be an addition salt with an acid or an addition salt with a base. For example, examples of acids in the addition salt of the heterocyclic compound of general formula (1) with an acid include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, phosphorous acid, nitrous acid, citric acid, formic acid, acetic acid, oxalic acid, maleic acid, lactic acid, tartaric acid, fumaric acid, benzoic acid, mandelic acid, cinnamic acid, pamoic acid, stearic acid, glutamic acid, aspartic acid, methanesulfonic acid, ethanedisulfonic acid, p-toluenesulfonic acid, salicylic acid, succinic acid, trifluoroacetic acid, etc. Examples of addition salts include hydrochloride, hydrobromide, hydroiodide, sulfate, sulfamate, phosphate, nitrate, phosphate, citrate, formate, acetate, oxalate, maleate, lactate, tartrate, fumarate, benzoate, mandelate, cinnamate, pamoate, stearate, glutamate, aspartate, methanesulfonate, ethanedisulfonate, p-toluenesulfonate, salicylate, succinate, and trifluoroacetate. On the other hand, examples of bases in addition salts of heterocyclic compounds of general formula (1) with a base include sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, ammonia, ethanolamine, and meglumine. Examples of base addition salts include sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, ethanolamine salt, and meglumine salt. 【0025】 Salts of heterocyclic compounds represented by the above general formula (1) exhibit excellent solubility in water and buffer solutions with a pH near neutral. Therefore, salts of heterocyclic compounds represented by the above general formula (1) can be dissolved at high concentrations in water and buffer solutions with a pH near neutral, thereby improving luminescence brightness. 【0026】 (Cytochrome P450) The aforementioned cytochrome P450 (CYP) is a group of reduced protoheme-containing proteins possessing monooxygenase activity. When carbon monoxide is passed through these proteins in a reduced state, their absorption spectrum changes, resulting in a difference spectrum (CO difference spectrum) with a maximum at 450 nm. The cytochrome P450 gene is known to be present in most organisms, with the exception of some bacteria such as Escherichia coli. Cytochrome P450 is known to be involved in various reactions, including hydroxylation, epoxidation, and demethylation, and its roles in vivo are diverse, including secondary metabolism, steroid hormone biosynthesis, xenobiotic metabolism, and hydrocarbon assimilation. For example, cytochrome P450 is involved in the metabolism of hydrophobic drugs, carcinogens, and other potentially toxic compounds and metabolites circulating in the blood. The liver is a major organ for xenobiotic metabolism, containing high levels of the most important CYP mixed-function oxidases. It is said that cytochrome P450 is involved in approximately 80% of drug metabolism reactions, and by measuring the activity of cytochrome P450, it is possible to evaluate drug side effects. 【0027】 The aforementioned cytochrome P450s are classified based on the identity of their amino acid sequences. As a general rule, if the amino acid sequences are 40% or more identical, they are classified into the same family, and if they are 55% or more identical, they are classified into the same subfamily, and a unique classification code is assigned. The classification code includes the CYP number representing the cytochrome P450, the family number, the subfamily number, and the gene number in that order, with the gene number assigned in the order of discovery. Examples of the cytochrome P450 subfamily (molecular species) include CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, and the like. Among these, CYP3A4, CYP2D6, CYP2C9, CYP2C8, and CYP1A2 are preferred from the viewpoint of their contribution to drug metabolism, with CYP3A4, CYP2D6, and CYP2C9 being even more preferred. These cytochrome P450 isoforms have a high contribution to drug metabolism and are particularly effective in evaluating drug metabolism. Furthermore, from the viewpoint of reactivity with heterocyclic compounds represented by the general formula (1) or their salts, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2E1, CYP3A4, and CYP3A5 are preferred, and CYP2A6, CYP2C8, CYP2C9, and CYP3A4 are more preferred. These cytochrome P450 molecular species exhibit high luminescence emission upon reaction with heterocyclic compounds represented by the general formula (1) or their salts, and can be detected with high sensitivity. 【0028】 In one preferred embodiment of the luminescence system of the present invention, the cytochrome P450 is present in the tissue of a living organism. In this case, luminescence originating from the cytochrome P450 in the tissue of a living organism is obtained. To detect luminescence originating from cytochrome P450 present in the tissue of a living organism, the heterocyclic compound represented by the general formula (1) or a salt thereof may be administered to the living organism and the light generated by the reaction may be detected, or the living organism may be collected and brought into contact with the heterocyclic compound represented by the general formula (1) or a salt thereof in a container such as a test tube and the light generated may be detected. 【0029】 In another preferred embodiment of the luminescence system of the present invention, the cytochrome P450 is present in the liver tissue of mammals. In this case, luminescence originating from the cytochrome P450 in the liver tissue of mammals is obtained. Generally, in mammals, cytochrome P450 is present in high concentrations in the liver tissue, so by detecting the luminescence originating from cytochrome P450 in the liver tissue, the drug metabolism capacity and side effects of the target mammal can be evaluated with high accuracy. To detect the luminescence originating from cytochrome P450 present in the liver tissue of mammals, the heterocyclic compound represented by the general formula (1) or a salt thereof may be administered to a mammal, delivered to the liver, and the light generated by the reaction with cytochrome P450 present in the liver tissue may be detected. Alternatively, liver tissue may be collected from a mammal and brought into contact with the heterocyclic compound represented by the general formula (1) or a salt thereof in a container such as a test tube, and the generated light may be detected. 【0030】 In another preferred embodiment of the luminescence system of the present invention, the cytochrome P450 is present in a container such as a test tube. In this case, since the luminescence derived from cytochrome P450 can be detected outside the body, the impact on living organisms can be reduced, and the absorption and scattering of light by components in the body (e.g., hemoglobin, oxyhemoglobin, water) can also be reduced, making it easier to detect the generated light. Furthermore, if necessary, the collected biological tissue can be purified to detect the luminescence derived from cytochrome P450 with greater accuracy. 【0031】 The heterocyclic compound represented by the above general formula (1) or its salt is present in a quantity of 1 mole to 1 × 10⁻¹⁶ of cytochrome P450 per mole. 5 It is preferable to use moles, 1 × 10 1 mol ~ 1 × 10 4 It is even more preferable to use moles. In this case, the amount of luminescence from the reaction between cytochrome P450 and the heterocyclic compound represented by general formula (1) or its salt increases depending on the amount of cytochrome P450, thereby improving the accuracy of the quantitative determination of cytochrome P450. 【0032】 (others) The luminescence system of the present invention may consist of a heterocyclic compound represented by the general formula (1) or a salt thereof, and cytochrome P450 as its components, but may also contain other components for purposes such as increasing the amount of luminescence or stabilizing the luminescence. Furthermore, it is sufficient that the heterocyclic compound represented by the general formula (1) or a salt thereof is present when reacting with cytochrome P450; for example, when administered in a container such as a test tube or into a living organism, it may be a precursor of the heterocyclic compound represented by the general formula (1) or a salt thereof. As a precursor of the heterocyclic compound represented by the general formula (1) or a salt thereof, R in general formula (1) may be used. 1 Ya R 2 Examples include, but are not limited to, compounds in which sugars, ATP, phospholipids, etc. are bound, and their salts. These precursors also include compounds in which sugars, ATP, phospholipids, etc. are bound. 1 Ya R 2 By being separated from the compound and changing into a heterocyclic compound represented by general formula (1) or a salt thereof, it reacts with cytochrome P450 and emits light. 【0033】 The luminescence system of the present invention preferably further includes a buffer. When a buffer is included, the pH of the luminescence system is easier to adjust, and stable luminescence is easier to obtain. Here, the pH of the luminescence system is preferably 4 to 10, and more preferably 6 to 8. Examples of buffers include potassium phosphate, Tris-HCl, glycine, and HEPES. 【0034】 The luminescence system of the present invention preferably further contains a surfactant. Examples of the surfactant include cationic surfactants, anionic surfactants, nonionic surfactants, and zwionic surfactants, with nonionic surfactants being preferred. Furthermore, poly(oxyethylene)octylphenyl ether (Triton X-100, etc.) is preferred as a nonionic surfactant. The surfactant content in the luminescence system is preferably in the range of 0.1 to 3% by mass, and more preferably in the range of 0.5 to 2% by volume. 【0035】 The luminescence system of the present invention preferably does not contain firefly luciferase. When the luminescence system does not contain firefly luciferase, luminescence due to the reaction between firefly luciferase and the heterocyclic compound represented by general formula (1) or its salt can be prevented, and only luminescence due to the reaction between cytochrome P450 and the heterocyclic compound represented by general formula (1) or its salt can be observed, thereby improving sensitivity to cytochrome P450. 【0036】 <Method for quantifying cytochrome P450> The present invention provides a method for quantifying cytochrome P450, characterized by reacting cytochrome P450 with a heterocyclic compound represented by the above general formula (1) or a salt thereof, measuring the amount of light emitted, and determining the amount of cytochrome P450 from the amount of light emitted. The present invention provides a simple and highly sensitive method for quantifying cytochrome P450. 【0037】 The method for quantifying cytochrome P450 according to the present invention may be performed in vivo or in vitro. When performed in vivo, the heterocyclic compound represented by the above general formula (1) or a salt thereof is administered to the body, and the administered heterocyclic compound of general formula (1) or a salt thereof reacts with cytochrome P450 in the body. By measuring the amount of light generated, the amount of cytochrome P450 in the body can be quantified. Furthermore, when performed in vitro, for example, a biotissue (particularly mammalian liver tissue) can be collected from a living organism, placed in a container, and a heterocyclic compound represented by the general formula (1) or a salt thereof can be added to the container. The added heterocyclic compound of general formula (1) or a salt thereof reacts with the cytochrome P450 in the biotissue in the container, and the amount of light generated can be measured to quantify the cytochrome P450 in the biotissue. 【0038】 The amount of light emitted by the reaction between the cytochrome P450 and the heterocyclic compound represented by the general formula (1) or its salt can be measured using a luminometer, image analyzer, scintillation counter, photointensifier tube photometer, photosensitive emulsion film, etc. 【0039】 <Applications of luminescence systems and cytochrome P450 quantitative analysis methods> The luminescence system of the present invention directly evaluates the activity of cytochrome P450, which is present in almost all living organisms, from bacteria and plants to mammals, and can therefore light-sens drug metabolism, hormone biosynthesis, fatty acid metabolism, and secondary metabolism in plants within these organisms. Furthermore, the non-invasive nature and deep visualization capabilities of bioluminescence imaging using heterocyclic compounds represented by the general formula (1) above, or their salts, enable in-vivo observation, which was previously difficult. Furthermore, since the luminescence system of the present invention does not require firefly luciferase and does not require the introduction of the luciferase gene, the activity evaluation of cytochrome P450 is simple, and it can be applied not only to experimental animals but also to the diagnosis of diseases and health conditions in humans. Its applications will be described in more detail below. 【0040】 (1) Bio-optical imaging Because the present invention is a luminescence system that does not require the introduction of genes such as the bioluminescent enzymes of fireflies, it can be applied not only to experimental animals but also to the diagnosis of diseases and health conditions in humans. 【0041】 (2) Diagnosis of disease When the activity (amount) of cytochrome P450 changes due to a disease, the luminescence system of the present invention can be used to measure the change in the amount of luminescence derived from cytochrome P450, thereby enabling the diagnosis of the disease. 【0042】 (3) Pharmacokinetic and safety evaluation Many drugs are known to be metabolized and excreted from the body by cytochrome P450. Therefore, determining which cytochrome P450 a novel drug is metabolized by and to what extent is an essential consideration in evaluating the safety of that drug. Since this metabolic process can be easily detected using the luminescence system of the present invention, the luminescence system of the present invention is useful for pharmacokinetic and safety evaluation. 【0043】 (4) Agriculture, forestry and fisheries sector (pesticides, variety improvement, increased production, etc.) In plants, cytochrome P450 plays a significant role in secondary metabolism. If the activity of cytochrome P450 can be easily visualized, screening becomes much easier during the process of improving plant varieties to produce those rich in specific nutrients. Therefore, the luminescence system of the present invention is useful in agriculture and forestry. In aquatic products, cytochrome P450 plays a significant role in secondary metabolism, and the luminescence system of the present invention can be utilized in the fisheries industry, including in the cultivation of aquatic products containing high levels of specific nutrients. Thus, in both agriculture and fisheries, the luminescence system of the present invention makes it possible to easily evaluate safety and other aspects, similar to human drug discovery, in the development of pesticides and antibiotics for livestock. 【0044】 (5) Environmental field (endocrine disruptors, environmental pollution, etc.) Fish are sometimes used to detect environmental pollution, including endocrine disruptors. In such cases, the activity of cytochrome P450 in fish is sometimes used as an indicator. Therefore, by using the luminescence system of the present invention to detect the activity of cytochrome P450 in fish, the luminescence system of the present invention can be effectively utilized for the detection of environmental pollution. [Examples] 【0045】 The present invention will be described in more detail below with reference to examples, but the present invention is not limited in any way to the following examples. 【0046】 <Tested heterocyclic compounds> In the following experiment, the heterocyclic compound represented by general formula (1) or its salt is defined as follows: [ka] The compound represented by (the deHCl type of "TokeOni" manufactured by Kurogane Kasei Co., Ltd.), or the following structural formula (1-2): [ka] The compound represented by (TokeOni, manufactured by Kurogane Kasei Co., Ltd.), or the following structural formula (1-3): [ka] The compound represented by (SeMpai, manufactured by Kurogane Kasei Co., Ltd.), or the following structural formula (1-4): [ka] Compounds represented by the following structural formulas (1-5): [ka] Compounds represented by the following structural formulas (1-6): [ka] Compounds represented by the following structural formulas (1-7): [ka] Compounds represented by the following structural formulas (1-8): [ka] The compound represented by [the formula shown] was used. The compounds of structural formulas (1-3) and (1-7) were synthesized by the method described in Japanese Patent Publication No. 6353751. Furthermore, the compounds shown in structural formulas (1-6) were synthesized using the method described in International Publication No. 2013 / 027770. Furthermore, the compound with structural formula (1-4) was synthesized by replacing the starting material compound having a naphthalene ring with a compound having a benzene ring, in the same synthesis method as the compound with structural formula (1-6). Furthermore, the compounds of structural formula (1-5) were synthesized by repeatedly performing the double bond formation step in the synthesis method described in Japanese Patent Publication No. 5464311. Furthermore, the compounds of structural formula (1-8) were synthesized in the same manner as described in the synthesis method of Japanese Patent No. 5464311, except that in the step of "Synthesis of Methyl Ester 1" in Example 1-3, "L-cysteine-S-trityl compound" was used instead of "D-cysteine-S-trityl compound". After synthesizing the L-isomer of the compound of structural formula (1-1), it was converted to hydrochloride (HCl) according to the synthesis method described in Japanese Patent No. 6011974. 【0047】 Also, for comparison, see the following chemical formula (a): [ka] This product uses natural firefly luciferin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), represented by [the formula shown]. 【0048】 <Measuring equipment> Furthermore, a luminometer (ATTO Corporation, "Luminescence Sensor AB2200") was used to measure the amount of luminescence. The measurement was performed by adding a heterocyclic compound represented by general formula (1) or a salt thereof, or firefly luciferin, and then recording the cumulative value of the luminescence over 30 seconds. 【0049】 (1) Observation of bioluminescence using cultured liver cells of arthropods, annelids, or mammals and cytochrome P450. (1-1) Arthropod and Annelida Extracts Each arthropod or annelid was homogenized in 0.5 mL of buffer (10 mM phosphate buffer (pH 7.4), 20% glycerol). The homogenates were centrifuged at 15,000 × g at 4°C for 15 minutes, and the supernatant was used as the extract for measurement. The solution composition used for luminescence measurement was 50 mM Tris / HCl, 20 mM KPB, 30 mM KCl, 8 mM MgCl2, 6% (v / v) glycerol, 1% (v / v) Triton X-100, and 200 μM of the compound with structural formula (1-1) [compound (1-1) in Figure 1] or 200 μM firefly luciferin. The results are shown in Figure 1. 【0050】 (1-2) Mammalian liver culture cells 150cm 2 After harvesting cultured rat, mouse, and human liver cells from cultured petri dishes, they were homogenized in 0.1 mL of buffer (10 mM phosphate buffer (pH 7.4), 20% glycerol). The homogenates were centrifuged at 15,000 × g at 4°C for 15 minutes, and the supernatant was used as the extract for measurement. The solution composition used for luminescence measurement was 50 mM Tris / HCl, 20 mM KPB, 30 mM KCl, 8 mM MgCl2, 6% (v / v) glycerol, 1% (v / v) Triton X-100, and 200 μM of the compound with structural formula (1-1) [compound (1-1) in Figure 2] or 200 μM firefly luciferin. The results are shown in Figure 2. 【0051】 Figure 1 shows that luminescence can be induced when the compound with structural formula (1-1) is added to extracts of arthropods and annelids. Furthermore, Figure 2 shows that luminescence can be induced by adding the compound with structural formula (1-1) to extracts of human, rat, and mouse liver cells. On the other hand, it was found that luminescence could not be detected when firefly luciferin was administered. 【0052】 (2) Observation of the decrease in luminescence due to cytochrome P450 inhibitors (2-1) Extracts of pill bugs and black flies Three pill bugs or five black flies were homogenized in 0.5 mL of buffer (10 mM phosphate buffer (pH 7.4), 20% glycerol). The homogenates were centrifuged at 15,000 × g at 4 °C for 15 minutes, and the supernatant was used as the extract for measurement. The solution composition used for the luminescence measurement was 50 mM Tris / HCl, 20 mM KPB, 30 mM KCl, 8 mM MgCl2, 6% (v / v) glycerol, 1% (v / v) Triton X-100, and 200 mM of the compound with structural formula (1-1). The results are shown in Figures 3 to 6. 【0053】 In the graphs shown in Figures 3 to 6, the luminescence value when the solvent DMSO is added is set to 100%, and the values ​​are shown as relative values. The inhibitor concentration was 2 mM in all cases. The amount of DMSO added was 2% in all cases. 【0054】 Table 1 shows the relationship between each inhibitor and its target cytochrome P450 (CYP). [Table 1] 【0055】 (2-2) Mouse liver extract Liver tissue was removed from C57BL6 mice after euthanasia, and homogenized with 3.0 mL of buffer (i) (10 mM phosphate buffer (pH 7.4), 20% glycerol) per mouse. The homogenates were centrifuged at 15,000 × g at 4°C for 15 minutes, and the supernatant was collected and filtered using a syringe filter with a pore size of 0.45 mm. Next, the filtrate was subjected to an anion exchange column (HiTrap Q FF: GE Healthcare). After equilibrating the column with buffer (i), the filtrate was loaded onto the column at a flow rate of 0.6 mL / min, and the pass-through fraction was collected. Next, this pass-through fraction was subjected to a hydroxyapatite column (Bio-Scale Mini CHT Type I cartridge: Bio-Rad). After column equilibration with buffer (i), the column was loaded at a flow rate of 0.6 mL / min, and the pass-through fraction was collected. The hydroxyapatite column was washed with 15 mL of buffer (i), and then eluted with buffer (ii) (150 mM phosphate buffer (pH 7.4), 20% glycerol). For luminescence measurement, a mixture of the pass-through fraction and the eluate fraction was used. The solution composition used for the luminescence measurement was 50 mM Tris / HCl, 65 mM PB, 30 mM KCl, 8 mM MgCl2, 6% (v / v) glycerol, 1% (v / v) Triton X-100, and 200 mM of the compound with structural formula (1-1). The results are shown in Figure 7. 【0056】 In the graph shown in Figure 7, the luminescence value when the solvent DMSO is added is set to 100%, and the values ​​are shown as relative values. The inhibitor concentration was 2 mM in all cases. The amount of DMSO added was 2% in all cases. 【0057】 Table 2 shows the relationship between each inhibitor and its target cytochrome P450 (CYP). [Table 2] 【0058】 Figures 2 to 6 show that when cytochrome P450 inhibitors were added to extracts derived from pill bugs or black houseflies, several inhibitors exhibited an inhibitory effect on luminescence. Furthermore, as shown in Figure 7, adding cytochrome P450 inhibitors to mouse liver extract also showed that some inhibitors inhibited luminescence. These results indicate that the luminescence of the compound with structural formula (1-1) is caused by the action of cytochrome P450. 【0059】 (2-3) Tests in mice (in vivo) Thirty minutes before the luminescence measurement, 100 μL of each inhibitor dissolved in DMSO was administered intraperitoneally to 6 to 10-week-old C57BL6 mice. Next, fifteen minutes before the luminescence measurement, the anesthetic pentobarbital was administered intraperitoneally to the mice at a dose of 65 mg per kg of body weight. Furthermore, five minutes before the luminescence measurement, 250 μL of the compound with structural formula (1-2) at 20 mM was administered intraperitoneally to the mice. The measurement was performed for 20 minutes using an image analyzer LS4000 (GE Healthcare Japan). The results are shown in Figure 8. 【0060】 Table 3 shows the relationship between each inhibitor and its target cytochrome P450 (CYP). [Table 3] 【0061】 Figure 8 shows that adding a cytochrome P450 inhibitor to mice also inhibited luminescence. These results indicate that the luminescence of the compound shown in structural formula (1-2) is caused by the action of cytochrome P450. 【0062】 (3) Examination of luminescence using cytochrome P450 For the cytochrome P450 (CYP) enzyme, we used human P450 enzyme from Corning. Furthermore, as a control, we used the membrane fraction of insect cells sold by Corning for control experiments. The value obtained by subtracting the measurement value from the CYP measurement value was used as the luminescence amount from the luminescence system consisting of CYP and each substrate. 【0063】 The CYP concentration during luminescence measurement is 100 pmol / mL. In addition, the other compositions of the solution used during luminescence measurement were 46 mM Tris / HCl, 17 mM phosphate buffer, 24 mM KCl, 6.4 mM MgCl2, 17 mM NaCl, 2% (v / v) Triton X-100, and a variable amount of luminescent substrate. The concentrations of the luminescent substrates are as follows: compound (1-1): 200 μM, compound (1-2): 800 μM, compound (1-3): 200 μM, compound (1-4): 100 μM, compound (1-5): 40 μM, compound (1-6): 100 μM, and compound (1-7): 200 μM. The results are shown in Figures 9 to 15. 【0064】 In the graphs shown in Figures 9 to 15, the luminescence values ​​when the compound with structural formula (1-3) is added are set to 100%, and the values ​​are shown as relative values. 【0065】 Figures 9 to 15 show that luminescence occurs when cytochrome P450 is combined with a heterocyclic compound represented by general formula (1) or a salt thereof. 【0066】 (4) Confirmation experiment using isomers Fifteen minutes before the luminescence measurement, 6 to 10-week-old C57BL6 mice were administered the anesthetic pentobarbital at a dose of 65 mg per kg of body weight intraperitoneally. Furthermore, five minutes before the luminescence measurement, 250 μL of either the compound with structural formula (1-2) (D-form) or the compound with structural formula (1-8) (L-form) was administered intraperitoneally to the mice at a 20 mM concentration. The measurement was performed for 20 minutes using an image analyzer LS4000 (GE Healthcare Japan). The results are shown in Figure 16. 【0067】 Figure 16 shows that the amount of luminescence is the same whether the compound with structural formula (1-2) (D-form) or the compound with structural formula (1-8) (L-form) is used. 【0068】 (5) Bioluminescence tests in insects (in vivo) Fifteen minutes before the bioluminescence measurement, 3 μL of a 20 mM compound with structural formula (1-2) was administered to 5-6 day old black flower fly larvae. The measurement was performed for 5 minutes using an image analyzer LS4000 (GE Healthcare Japan), and images were acquired. The results are shown in Figure 17. In Figure 17, the white areas indicate the bioluminescent sites. 【0069】 Figure 17 shows that light emission occurs when black mallow fly larvae (containing cytochrome P450) are combined with a heterocyclic compound represented by general formula (1) or a salt thereof. 【0070】 (6) Luminescence test in a mouse model of drug-induced liver injury induced by carbon tetrachloride administration A chronic liver disease model mouse was created by administering carbon tetrachloride, diluted fivefold with olive oil, intraperitoneally twice a week for four weeks at a dose of 3 mL / kg to seven-week-old C57BL6 mice. The control group was administered the same amount of olive oil. Fifteen minutes before the luminescence measurement, the anesthetic pentobarbital was administered intraperitoneally to the mice at a dose of 65 mg per kg of body weight. Furthermore, five minutes before the luminescence measurement, 250 μL of the compound with structural formula (1-2) at 20 mM was administered intraperitoneally to the mice, and the measurement was performed. The results are shown in Figure 18. 【0071】 Figure 18 shows that the luminescence output is reduced in the chronic liver disease model mice compared to the control group of mice. This indicates that drug-induced liver injury reduced the amount of cytochrome P450 in the mice. 【0072】 Regarding literature on drug-induced toxic liver injury, for example, the Journal of the Japanese Society of Internal Medicine, 84, 188-193 (1995), discloses that administration of carbon tetrachloride reduces the amount of cytochrome P450 in the liver, which was confirmed to be consistent with the above test results. 【0073】 (7) Luminescence test in a mouse model of non-alcoholic fatty liver disease Seven-week-old C57BL6 mice were given a choline-deficient CDAHFD high-fat diet as ad libitum for eight weeks. The control group was given the same amount of normal diet as ad libitum. Fifteen minutes before the luminescence measurement, the anesthetic pentobarbital was administered intraperitoneally to the mice at a dose of 65 mg per kg of body weight. Furthermore, five minutes before the luminescence measurement, 250 μL of the compound with structural formula (1-2) at 20 mM was administered intraperitoneally to the mice, and the measurement was performed. The results are shown in Figure 19. 【0074】 Figure 19 shows that the luminescence level is increased in non-alcoholic fatty liver disease (NADL) model mice compared to the control group. This indicates that NDL increases cytochrome P450 levels in the mice. 【0075】 Regarding literature on non-alcoholic steatohepatitis, for example, Weltman MD et al. (1998), “Hepatic cytochrome P450 2E1 is increased in patients with nonalcoholic steatohepatitis”, Hepatology, 27(1):128-33, discloses that CYP2E1 expression is induced in patients with non-alcoholic steatohepatitis, which was confirmed to be consistent with the results of the above-mentioned study. [Industrial applicability] 【0076】 As described above, the light-emitting system of the present invention can be used in bio-optical imaging, disease diagnosis, pharmacokinetic and safety evaluation, agriculture, forestry and fisheries (pesticides, crop improvement, increased production, etc.), and environmental fields (endocrine disruptors, environmental pollution, etc.). Furthermore, the luminescence system of the present invention can be used in various kits such as glucose monitoring kits, cardiac markers, infectious disease testing kits, pregnancy and infertility testing kits, blood glucose, blood gas and electrolyte test kits, tumor / cancer markers, urine test kits, cholesterol test kits, immunotest kits, pharmacokinetic and safety evaluation kits, and food toxicity evaluation kits. Furthermore, the light-emitting system of the present invention can also be used in the fields of medical research, evolutionary and life sciences research, and astrobiology (such as microgravity science experiments using the International Space Station (ISS) and the Lunar Orbital Platform Gateway).

Claims

[Claim 1] The following general formula (1): 【Chemistry 1】 [In the formula, R １ is hydrogen or an alkyl group having 1 to 4 carbon atoms. R ２ NR ４ ２ Or OH, where R ４ Each is independently hydrogen or an alkyl group having 1 to 6 carbon atoms, and NR ４ ２ The two R's ４ They may be joined to each other to form a ring. A is given by the following general formula (2) or (3): 【Chemistry 2】 It is represented as, R in the above general formula (2) ３ is each independently CR ５ or N, and R3 in the above general formula (3) is each independently CR5, where R ５ is each independently hydrogen, an alkyl group having 1 to 8 carbon atoms or an alkenyl group having 2 to 8 carbon atoms. A heterocyclic compound represented by [n is 1, 2, or 3] or a salt thereof, It contains cytochrome P450, excluding those present in human liver tissue, and does not contain firefly luciferase. A light-emitting system characterized by the reaction of a heterocyclic compound represented by the above general formula (1) or a salt thereof with the cytochrome P450 to produce light. [Claim 2] The following general formula (1): 【Transformation 3】 [In the formula, R １ is hydrogen or an alkyl group having 1 to 4 carbon atoms. R ２ NR ４ ２ Or OH, where R ４ Each is independently hydrogen or an alkyl group having 1 to 6 carbon atoms, and NR ４ ２ The two R's ４ They may be joined to each other to form a ring. A is given by the following general formula (2) or (3): 【Chemistry 4】 It is represented as, In the above general formula (2), R ３ Each of them is independently CR ５ Or N, and in the above general formula (3), R 3 is independently CR 5, where R ５ These are, independently, hydrogen, an alkyl group having 1 to 8 carbon atoms, or an alkenyl group having 2 to 8 carbon atoms. A heterocyclic compound or salt thereof represented by [n is 1, 2, or 3], A luminescent system characterized by containing cytochrome P450, excluding those present in human liver tissue. [Claim 3] The following general formula (1): 【Transformation 5】 [In the formula, R １ is hydrogen or an alkyl group having 1 to 4 carbon atoms. R ２ NR ４ ２ Or OH, where R ４ Each is independently hydrogen or an alkyl group having 1 to 6 carbon atoms, and NR ４ ２ The two R's ４ They may be joined to each other to form a ring. A is given by the following general formula (2): 【Transformation 6】 It is represented as, R ３ Each of them is independently CR ５ Or N, where R ５ These are, independently, hydrogen, an alkyl group having 1 to 8 carbon atoms, or an alkenyl group having 2 to 8 carbon atoms. A heterocyclic compound or salt thereof represented by [n is 1, 2, or 3], A luminescent system characterized by containing cytochrome P450, excluding those present in human liver tissue. [Claim 4] The luminescence system according to any one of claims 1 to 3, wherein the cytochrome P450 is present in the tissue of a living organism. [Claim 5] The luminescence system according to any one of claims 1 to 4, wherein the cytochrome P450 is present in mammalian liver tissue. [Claim 6] Cytochrome P450, excluding that present in human liver tissue, is provided with the following general formula (1): 【Transformation 7】 [In the formula, R １ is hydrogen or an alkyl group having 1 to 4 carbon atoms. R ２ NR ４ ２ Or OH, where R ４ Each is independently hydrogen or an alkyl group having 1 to 6 carbon atoms, and NR ４ ２ The two R's ４ They may be joined to each other to form a ring. A is given by the following general formula (2) or (3): 【Transformation 8】 It is represented as, In the above general formula (2), R ３ Each of them is independently CR ５ Or N, and in the above general formula (3), R 3 is independently CR 5, where R ５ These are, independently, hydrogen, an alkyl group having 1 to 8 carbon atoms, or an alkenyl group having 2 to 8 carbon atoms. A heterocyclic compound represented by [n is 1, 2, or 3] or a salt thereof is reacted without firefly luciferase, and the amount of light emitted by the reaction between the cytochrome P450 and the heterocyclic compound represented by the general formula (1) or a salt thereof is measured. A method for quantifying cytochrome P450, characterized by measuring the amount of cytochrome P450 from the amount of light emitted. [Claim 7] Cytochrome P450, excluding that present in human liver tissue, comprising the following general formula (1): 【Chemistry 9】 [In the formula, R １ is hydrogen or an alkyl group having 1 to 4 carbon atoms. R ２ NR ４ ２ Or OH, where R ４ Each is independently hydrogen or an alkyl group having 1 to 6 carbon atoms, and NR ４ ２ The two R's ４ They may be joined to each other to form a ring. A is given by the following general formula (2) or (3): 【Chemistry 10】 It is represented as, In the above general formula (2), R ３ Each of them is independently CR ５ Or N, and in the above general formula (3), R 3 is independently CR 5, where R ５ These are, independently, hydrogen, an alkyl group having 1 to 8 carbon atoms, or an alkenyl group having 2 to 8 carbon atoms. A method for generating light, characterized by reacting a heterocyclic compound represented by [n is 1, 2, or 3] or a salt thereof without firefly luciferase, and generating light through the reaction of the cytochrome P450 with the heterocyclic compound represented by the above general formula (1) or a salt thereof.

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