Method for measuring one or more biomarkers

Abstract

Provided are: a method for measuring one or more biomarkers to simply and accurately diagnose diseases associated with changes in the activities of cytochrome P450 metabolic enzymes (hereinafter referred to as "P450s"); and a method for detecting the one or more biomarkers. The methods focus on luminescence-based detection of the enzymatic activities of P450s, and involves reacting one or more P450s present in a sample collected in a low- or non-invasive manner, such as serum or urine, with a precursor of a luminescent / light-emitting substrate, metabolizing a luminescent substrate converted from the precursor to generate luminescence, and measuring the generated luminescence. Since many molecular species of P450s are known, the activities of different molecular species of P450s can be measured for each disease on the basis of luminescence intensity, and the activity pattern of each P450 can be analyzed to detect one or more biomarkers.

Description

Method for measuring biomarker

[0001] The present invention relates to a method for measuring a biomarker for the examination of diseases in which cytochrome P450 activity fluctuates.

[0002] This application claims the priority of Japanese Patent Application No. 2024-185688, which is hereby incorporated by reference.

[0003] Organs such as the liver and large intestine are organs in which patients are unlikely to experience subjective symptoms from the early to mid-stage of cancer development. Therefore, in order to maintain the liver and large intestine in a good state before and after treatment, it is important to undergo regular medical check-ups. Generally, cancer diagnosis is made based on the subjective comprehensive judgment of a doctor through clinical symptoms, findings from ultrasonic examinations and X-ray CT examinations, and hematological examinations. In addition, pathological examinations of hepatocytes and large intestine epidermal cells by biopsy are the most accurate diagnostic methods, but they impose a large burden on patients as a daily diagnostic technique. Therefore, a minimally invasive biomarker that does not involve pain or risk is required.

[0004] In the current medical field, omics analysis is being carried out to search comprehensively for biological molecule information such as genomes, using proteins, nucleic acids, lipids, etc. specific to cancer cells as cancer cell biomarkers. However, omics analysis has problems such as the need to identify specific biological molecule components of various cancer cells and the need for a great deal of effort for such specific analysis. Therefore, a biomarker that can be applied to simpler tests is required.

[0005] On the other hand, neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease are increasing rapidly in an aging society, and these diseases cause memory loss, personality breakdown, and loss of functions necessary for social life. For neurodegenerative diseases as well, a minimally invasive biomarker that does not involve pain or risk and can be applied to simpler tests is required.

[0006] In recent years, attention has been focused on the relationship between the enzymatic activity of cytochrome P450 (hereinafter sometimes simply referred to as "P450" or "CYP"), a metabolic enzyme, and disease. The present inventors' group has developed a method to measure the activity of multiple types of P450 using an oxygen sensor containing a ruthenium complex to obtain metabolic patterns as biomarkers, and has disclosed that the toxicity (mutagenicity) of compounds taken into the subject's body can be predicted based on the obtained metabolic patterns (Patent Document 1). The P450 activity measurement system for metabolic patterns disclosed in Patent Document 1 utilizes the fluorescence phenomenon caused by the ruthenium complex in response to a decrease in oxygen partial pressure.

[0007] P450 (CYP) enzymes are membrane-bound heme proteins distributed across a wide range of organisms, from microorganisms to plants and animals, playing crucial roles in drug detoxification, cellular metabolism, and homeostasis. In mammals in particular, P450 is involved in cholesterol synthesis, steroid hormone synthesis, lipid-soluble substance metabolism, and xenobiotic metabolism. The present inventors disclose a method for detecting biomarkers for inflammatory diseases, which includes detecting the fluorescence pattern by reacting multiple types of P450 with a fluorescent substrate in the presence of a sample and measuring the fluorescence emitted by the fluorescent substance converted from the fluorescent substrate (Patent Document 2). This method involves competitively reacting serum collected from patients and healthy individuals with artificially produced P450 and a fluorescent substrate in vitro, and comparing the resulting changes in fluorescence values ​​to distinguish between healthy individuals and patients.

[0008] Furthermore, the inventors obtained enzyme reaction inhibition patterns of substrates specifically affected by multiple types of P450, and investigated whether these enzyme reaction inhibition patterns could be used as disease biomarkers by performing characteristic analysis by comparing the enzyme reaction inhibition patterns in a system containing a sample with those without a sample. The fluorescence pattern of P450 activity was analyzed as the enzyme reaction inhibition pattern (Patent Document 3). However, the fluorescence method requires excitation light energy, resulting in background noise during measurement. Additionally, the need for an excitation light source leads to interference phenomena due to combined fluorescence, among other problems. The development of a more useful method than analyzing fluorescence patterns is desired.

[0009] Japanese Patent No. 4899050, Published Patent Application No. 2016-214127 (Japanese Patent No. 6501607), Published Patent Application No. 2021-159012 (Japanese Patent No. 7551096)

[0010] The present invention aims to provide a method for measuring and detecting a biomarker for easily and accurately diagnosing diseases involving fluctuations in P450 activity.

[0011] The inventors, after diligent research to solve the above problems, focused on detecting P450 enzyme activity by luminescence. They discovered that P450 activity can be measured by reacting one or more types of P450 in a minimally invasively collected sample, such as serum or urine, with a luminescent substrate precursor, and measuring the luminescence emitted when the luminescent substrate converted from the luminescent substrate precursor is metabolized. This led to the completion of the present invention. Since there are many molecular species of P450, the inventors have completed the invention by measuring the luminescence amount of P450 activity of various molecular species for each disease, analyzing the P450 activity pattern, and detecting biomarkers.

[0012] In other words, the present invention comprises the following: 1. A measurement method for testing diseases in which cytochrome P450 activity fluctuates, characterized by reacting one or more types of cytochrome P450 in a sample taken from a subject with a luminescent substrate precursor, and measuring the luminescence emitted by metabolizing the luminescent substrate converted from the luminescent substrate precursor. 2. The measurement method according to item 1 above, wherein the disease in which cytochrome P450 activity fluctuates is a disease in which cytochrome P450 activity fluctuates due to inflammation. 3. The measurement method according to item 1 above, wherein the luminescent substrate precursor is a luciferin precursor. 4. The measurement method according to item 1 above, wherein the luminescent substrate is luciferin, and the luminescence is luminescence due to the action of luciferin and luciferase. 5. The measurement method described in item 1 above, wherein the cytochrome P450 comprises one or more selected from CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, CYP3A7, CYP4F2, CYP4F3A, CYP4F3B, CYP4F12, and CYP19. 6. The measurement method described in item 1 above, wherein the specimen collected from the subject is a minimally invasive specimen. 7. The measurement method described in item 1 above, wherein the minimally invasive specimen is selected from blood-derived components, urine, saliva, sputum, and sweat. 8. A method for detecting a disease biomarker, comprising detecting the luminescence patterns of one or more types of cytochrome P450 by reacting one or more types of cytochrome P450 in a sample taken from a subject with a luminescent substrate precursor, and measuring the luminescence emitted by metabolizing the luminescent substrate converted from the luminescent substrate precursor. 9. The detection method according to item 8 above, wherein the disease is a disease in which the activity of one or more types of cytochrome P450 is expected to fluctuate. 10. An analytical method for testing a disease in which cytochrome P450 activity fluctuates due to inflammation, comprising the following steps: (1) a step of detecting a luminescence pattern in a sample taken from a subject by the measurement method described in any one of items 1 to 7 above; (2) a step of analyzing the overlap between the luminescence pattern detected in step (1) and a luminescence pattern previously created from a sample of a disease in which cytochrome P450 activity fluctuates.11. The analytical method described in paragraph 10 above, wherein the disease in which cytochrome P450 activity fluctuates due to inflammation is one of the diseases selected from cancer, neurological diseases, gastrointestinal diseases, respiratory diseases, and metabolic diseases.

[0013] A measurement method that involves reacting one or more types of P450 in a sample taken from a subject with a luminescent substrate precursor, and measuring the luminescence emitted when the luminescent substrate converted from the luminescent substrate precursor is metabolized, makes it possible to easily and accurately detect new biomarkers for diseases in which P450 activity fluctuates. While fluorescence methods require excitation light energy, resulting in background noise during measurement, and require an excitation light source, leading to interference phenomena due to combined fluorescence, this method of measuring luminescence does not require an excitation light source. Therefore, the method of the present invention allows for measurement under conditions with very low background noise. This enables accurate measurement results even from small amounts of sample, allowing for screening of multiple types of P450 and easy detection of biomarkers related to diseases in which P450 activity fluctuates. Thus, for diseases in which P450 activity fluctuates, this method allows for minimally invasive testing with minimal burden on the subject, such as for diseases in which cytochrome P450 activity fluctuates due to inflammation, and facilitates appropriate monitoring of the patient's condition.

[0014] The following shows the results of confirming the rate of change in activity for three molecular species, CYP1A1, CYP1A2, and CYP1B1, in serum samples from breast cancer patients and healthy individuals. (Example 1) The Receiver Operating Characteristic Curve (ROC curve) created from the results of confirming the rate of change in activity for three molecular species, CYP1A1, CYP1A2, and CYP1B1, in breast cancer patients is shown. (Example 2) The results of confirming the rate of change in activity for three molecular species, CYP1A1, CYP1A2, and CYP1B1, in serum samples from pancreatic cancer patients and healthy individuals are shown. (Example 3) The ROC curve created from the results of confirming the rate of change in activity for three molecular species, CYP1A1, CYP1A2, and CYP1B1, in pancreatic cancer patients is shown. (Example 4) The results of confirming the rate of change in activity for three molecular species, CYP1A1, CYP1A2, and CYP1B1, in urine samples from patients with depression and healthy individuals are shown. (Example 5) The ROC curve created from the results of confirming the rate of change in activity of two molecular isoforms, CYP1A1 and CYP1A2, in patients with depression is shown. (Example 5)

[0015] Cytochrome P450 (P450, CYP) comprises numerous molecular species with different substrate specificities. P450s are broadly classified into three groups (1, 2, and 3), and each of these groups is further classified into several subfamilies (A to E). Most P450s function as monooxygenases, catalyzing reactions such as substrate hydroxylation, epoxidation, demethylation, and NO synthesis. The major reaction involving P450s, hydrocarbon oxidation, is catalyzed by the dormant oxidized form of P450 (Fe 3+ The substrate binds to the protein, and generally utilizes electrons supplied from NADPH via electron transfer proteins to form a reduced form (Fe 2+ It begins with a change (one-electron reduction) to the heme of reduced P450. An oxygen molecule (O2) immediately coordinates to the heme, but the second one-electron reduction produces superoxide (O2 - ) from peroxide (O2 2- Auto-oxidation is prevented by the change to ). Subsequently, the proton (H + Through interaction with ), it forms an oxidatively active species, and returns to its oxidized form by dissociating with the product obtained by adding a single atom oxygen to the substrate.

[0016] In this invention, the type of P450 to be measured is not particularly limited as long as it has the above-mentioned characteristics. For example, in a disease where it is unclear whether or not P450 activity fluctuates, when attempting to detect a biomarker related to P450, it becomes possible to comprehensively measure and screen the P450 activity of all molecular species and easily detect the biomarker. For the reasons above, P450 can be arbitrarily selected, but it is preferable to select, for example, drug-metabolized P450s. Specifically, an appropriate P450 can be selected from the 57 currently known P450s. For example, 23 P450s can be listed, consisting of CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP2R1, CYP2W1, CYP3A4, CYP3A5, CYP4F2, CYP4F12, CYP4X1, CYP17A1, CYP27A1, and CYP51A1. An appropriate P450 can be selected from these. Examples include one or more selected from CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, CYP3A7, CYP4F2, CYP4F3A, CYP4F3B, CYP4F12, and CYP19.

[0017] In this specification, when P450 is used as a reagent, any P450 that is stable in vitro and can react with a luminescent substrate precursor or luminescent substrate is acceptable. P450 can be prepared by genetic engineering, for example, by the method described in Patent Document 3. The prepared P450 is preferably of human origin, but may also be derived from mammals such as monkeys, cattle, pigs, sheep, dogs, cats, mice, rats, guinea pigs, rabbits, and goats.

[0018] In this specification, the measurement of luminescent substances refers to measurement methods that utilize bioluminescence. These methods involve reacting P450 with a luminescent substrate precursor, and measuring the luminescence emitted when the luminescent substrate converted from the precursor is metabolized. Specifically, a luciferin precursor can be reacted with P450 as the luminescent substrate precursor, and the resulting luciferin can be used as the "luminescent substrate." When the luminescent substrate luciferin is reacted with luciferase, the luciferase oxidizes it to produce oxyluciferin, and the simultaneous emission of photons is observed at this time. All luciferases are known to catalyze this luminescence reaction. On the other hand, it has also become clear that luciferin substrates have diverse structures (James Inglese et al., High-throughput screening assays for the identification of chemical probes, Net. Chem. Biol, 2007 Aug; 3(8): 466-79). For example, some luciferin precursors do not emit light in their original state because they are not metabolized by the luciferase enzyme. On the other hand, these luciferin precursors react with P450 to transform into luciferin, a luminescent substrate, which then reacts with luciferase to become luminescent. In this invention, P450 activity can be measured from the amount of luminescence emitted by this principle. This makes it possible to detect biomarkers for diseases in which P450 activity fluctuates, and furthermore, by measuring the amount of luminescence for one or more types of P450 and detecting the luminescence pattern, it is possible to detect biomarkers for diseases.

[0019] Regarding luciferin precursors, luciferin precursors with diverse chemical structures have already been developed, and it has been reported that the reaction characteristics of luciferin precursors with P450 enzymes are also diverse. In the method of the present invention, the usable luciferin precursor is not particularly limited, as long as it is a luciferin precursor that can react individually with each P450 molecular species, but an example is luciferin Multi-CYP (Promega, Inc., USA). The aforementioned luciferin Multi-CYP is a substrate with mixed specificity that reacts with at least 21 different P450 molecular species. Any luciferin precursor reagent developed in the future that can react individually with each P450 molecular species will also be included in the present invention.

[0020] The principle of the measurement method of the present invention is illustrated by the following example. For instance, the methoxy group of luciferin Multi-CYP (Promega, USA), used as a luminescent substrate precursor, is demethylated by P450, converting it to the luminescent substrate luciferin. Subsequently, luciferin reacts with ATP, Mg + Furthermore, it is metabolized and luminescent by specifically reacting with luciferase in the presence of O2.

[0021]

[0022] As the most widely used bioluminescence system in the field of bioluminescence imaging (BLI), the firefly luciferin-luciferase system has been extensively studied over the past few decades. 2+ With the help of [unclear], firefly luciferase (Fluc) catalyzes the reaction between D-luciferin (D-LH2) and adenosine triphosphate (ATP) to produce the intermediate D-LH2-AMP complex. Subsequently, D-LH2-AMP is oxidized and decarboxylated to produce oxyluciferin, an unstable high-energy state. Oxyluciferin emits light during the relaxation process as it transitions to the ground state. This series of processes is influenced by many factors, including pH, solvent polarity, and the enzyme microenvironment. The wavelength of the light produced by this series of reactions is variable under different catalytic conditions, with the maximum emission wavelength ranging from 530 nm to 640 nm.

[0023] The luciferase used in this invention may be commercially available or can be produced, for example, by genetic engineering. The P450 produced is preferably of human origin, but may also be a luciferase derived from mammals such as monkeys, cattle, pigs, sheep, dogs, cats, mice, rats, guinea pigs, rabbits, or goats. When produced by genetic engineering, although not particularly limited, a firefly luciferase expression plasmid can be used, for example, by inserting firefly-derived luciferase cDNA downstream of the T7 promoter in Biodynamics' expression plasmid pETIA. After culturing E. coli transformed with this plasmid, the luciferase in the E. coli can be affinity-purified using the His tag sequence added to the C-terminus.

[0024] The measurement method of the present invention has the advantage of higher detection sensitivity compared to conventional fluorescence measurement methods. Unlike fluorescence, luminescence does not require excitation light energy, so the background noise during measurement is very low. Therefore, it is expected that accurate measurement results can be obtained even from small amounts of sample. Removing the excitation light source also prevents interference phenomena caused by complex fluorescence. Consequently, the luminescence measurement method of the present invention enables highly sensitive measurements despite having a significantly weaker signal intensity compared to fluorescence measurement methods. Furthermore, since luminescence occurs only in an environment where all the necessary substances for the luminescence phenomenon are provided, low background noise and highly sensitive detection are possible.

[0025] By detecting luminescence patterns from multiple types of P450 in a sample taken from the subject, and analyzing the overlap between the detected luminescence patterns and luminescence patterns previously created from samples of diseases with fluctuating P450 activity, it is possible to perform tests on the subject's disease, such as diagnosis, monitoring of the disease's progression, and confirmation of treatment effectiveness.

[0026] According to the method of the present invention, the presence or absence of a disease can be determined by comparing the emission patterns of multiple types of P450. For example, the presence or absence of cancer or neurodegenerative disease can be determined by comparing the emission patterns of multiple types of P450 in specimens of cancer and specimens of neurodegenerative disease. Furthermore, by accumulating a large amount of data, a trained classification model can be created, enabling even more detailed disease classification.

[0027] In the present invention, any means may be used to detect the amount of luminescence obtained from multiple types of P450 in the presence of a sample, for example, when performing screening. For example, in each compartment of an array having multiple compartments, a reaction can be carried out between a sample and a luciferin precursor that reacts with one type of P450, and the amount of luminescence emitted from the luciferin can be measured. For example, in each compartment of an array having multiple compartments, a reaction can be carried out between one type of P450 and a luciferin precursor that reacts with the said P450 in the presence of a sample, and a change in the amount of luminescence emitted from the luciferin can be detected. A compartment refers to a closed area that does not come into contact with other samples or luciferin precursors, and is also called a reaction vessel. An array having multiple compartments is a device having multiple reaction vessels, and examples of such devices include plates and disks.

[0028] For example, the following explanation will use a plate with multiple wells as an array. Each well on the plate contains one type of P450 and one type of luciferin precursor, so that the plate as a whole carries multiple types of P450. By placing P450, luciferin precursor, and a sample in the wells, a luminescence competition assay can be performed. For example, by comparing the luminescence in a well to which a sample has been added with the luminescence in a well to which no sample has been added, changes in luminescence due to the sample can be detected. If a substance with affinity for P450 is present in the sample, the activity of P450 will decrease and the amount of luminescence will decrease. If a substance that can enhance the activity of P450 is present in the sample, the activity of P450 will increase and the amount of luminescence will increase. Such changes in P450 activity can be detected by the amount of luminescence to obtain a luminescence pattern, and this luminescence pattern can be used as a biomarker for disease.

[0029] The luminescence pattern in this invention is based on the activity patterns of multiple types of P450, and can be comprehensively patterned by detecting the activity of multiple types of P450 through luminescence. The luminescence pattern in this invention may be created using any measurement and calculation method. For example, the luminescence pattern in this invention can also be constructed by identifying P450 types that show characteristic changes in luminescence intensity in the presence of a sample compared to the absence of a sample. Alternatively, the luminescence pattern in this invention can also be constructed by identifying P450 types whose luminescence intensity decreases in the presence of a sample compared to the absence of a sample, P450 types whose luminescence intensity remains unchanged, and P450 types whose luminescence intensity increases. The luminescence pattern may be constructed not only by identifying whether or not there is a change in the luminescence intensity of each P450, but also by identifying the degree of change in the luminescence intensity of each P450. The inhibition rate of the activity of each P450 by the sample can also be calculated from the luminescence intensity values, and the pattern of the inhibition rates can be used as the luminescence pattern.

[0030] The present invention also includes a method for testing for diseases, comprising the following steps: (1) detecting a luminescence pattern in a subject's sample using the above-mentioned method for detecting a biomarker for a disease; (2) determining a specific disease by analyzing the overlap between the luminescence pattern detected in step (1) and a luminescence pattern previously created from a sample of a specific patient.

[0031] In the disease testing method of the present invention, first, in step (1), a luminescence pattern is obtained using a sample from the subject by a biomarker detection method for a specific disease. By confirming whether the luminescence pattern obtained from the subject's sample overlaps with a luminescence pattern for a sample from a patient with a specific disease that has been prepared in advance, a determination of the specific disease can be made. The pre-prepared luminescence pattern serves as a standard for the testing method of the present invention. It is preferable to prepare the luminescence patterns for samples from patients with a specific disease in advance using the biomarker detection method for a specific disease of the present invention. For example, luminescence patterns for each type of specific disease, and luminescence patterns for each phase or stage of the same specific disease can be prepared in advance. It is also preferable to obtain the luminescence pattern for a sample from a healthy person in advance using the biomarker detection method for a specific disease of the present invention. By using such luminescence patterns as a standard, it is possible to determine the type of a specific disease, and the phase or stage of the same specific disease. Examples of stages of a specific disease include the initial stage, active stage, and remission stage. The testing method of the present invention makes it possible not only to diagnose the onset of a specific disease, but also to monitor the condition of patients with a specific disease, evaluate the effectiveness of treatment, and diagnose the prognosis. For example, if a particular disease is a chronic inflammatory disease (preferably inflammatory bowel disease), the subject is determined to have a chronic inflammatory disease (preferably inflammatory bowel disease) when a luminescence pattern showing elevated activity of at least CYP2B6, CYP1A2, and CYP2C19 is observed.

[0032] Diseases to which the measurement method of the present invention can be applied include diseases in which P450 activity fluctuates. Here, "diseases in which P450 activity fluctuates" is not limited to diseases in which P450 activity is known to fluctuate at the time of measurement, but may also include diseases in which P450 activity is predicted to fluctuate, or diseases in which it is unclear whether P450 activity fluctuates but it is necessary to determine this. As described above, the measurement method of the present invention is applicable to screening that comprehensively measures the P450 activity of all molecular species when attempting to detect biomarkers related to P450. According to the method of the present invention, the presence or absence of a disease can be determined by comparing the luminescence patterns of multiple types of P450.

[0033] Diseases in which P450 activity fluctuates include, for example, diseases in which P450 activity fluctuates due to inflammation. This is not limited to so-called inflammatory diseases, but encompasses all diseases involving inflammation. Diseases involving inflammation include chronic inflammatory diseases and acute inflammatory diseases, but chronic inflammatory diseases are preferred. Inflammatory diseases can be any inflammatory disease that can be diagnosed in the medical field, and are not particularly limited, and include, for example, cancer, neurological diseases, digestive system diseases, respiratory system diseases, metabolic diseases, etc. Examples include cancer, neurological diseases, digestive system diseases, respiratory system diseases, and metabolic diseases. Specific examples of chronic inflammatory diseases in the present invention include inflammatory bowel disease (IBD) (ulcerative colitis, Crohn's disease, etc.), rheumatoid arthritis, systemic lupus erythematosus, glomerulonephritis (nephrotic syndrome (idiopathic nephrotic syndrome, minimal change nephropathy, etc.)), multiple sclerosis, primary biliary cirrhosis, primary sclerosing cholangitis, myasthenia gravis, idiopathic sprue, sarcoidosis, Reiter's syndrome, type 1 diabetes mellitus, Harada's disease, Behçet's disease, Sjögren's syndrome, mixed connective tissue disease, Graves' disease, chronic thyroiditis, autoimmune hematological disorders (hemolytic anemia, irreversible anemia, idiopathic thrombocytopenia, etc.), chronic hepatitis B, chronic hepatitis C, chronic active EB virus infection, psoriasis-like arthritis, and inflammatory skin diseases (lichen planus, pemphigus, bullous pemphigoid, epidermolysis bullosa, alopecia areata, etc.).

[0034] Cancers include, in addition to those listed above, breast cancer, pancreatic cancer, liver cancer, colorectal cancer, stomach cancer, lung cancer, kidney cancer, uterine cancer, and bile duct cancer, as well as leukemia, malignant lymphoma, brain tumors, neuroblastoma, retinoblastoma, kidney tumors, liver tumors, and sarcomas such as osteosarcoma. Neurological diseases include, in addition to those listed above, Parkinson's disease, Alzheimer's disease (which impairs cognitive function), Lewy body dementia, and corticobasal degeneration, as well as Parkinson's disease and Parkinsonian syndromes (multiple system atrophy, progressive supranuclear palsy, etc.) (which impair motor function), amyotrophic lateral sclerosis (which causes muscle weakness), spinocerebellar degeneration and spastic paraplegia (which impair balance). Digestive system diseases include, in addition to those listed above, cirrhosis of the liver. Respiratory system diseases include, in addition to those listed above, chronic obstructive pulmonary disease (COPD). Metabolic diseases include, in addition to those listed above, type 1 diabetes and type 2 diabetes.

[0035] The specimen used in the present invention is not particularly limited as long as it is collected from a subject, however, it is preferable that the specimen collected from the subject be a minimally invasive specimen that is less burdensome to the subject. Examples of minimally invasive specimens include blood-derived components, urine, saliva, sputum, sweat, etc. Specifically, blood-derived components can be selected from whole blood, plasma, and serum, with serum being preferred. The collected specimen can be pre-treated as appropriate before performing the test. For example, when human serum is used as a specimen, the serum can be diluted as appropriate, for example, 10 to 100 times, preferably 30 to 50 times, and more preferably 40 times. The solution used for dilution is not particularly limited as long as it can achieve the objectives of the present invention. In pharmacokinetic studies involving P450s (evaluation of the in vivo dynamics of pharmaceutical and food components through P450 metabolism), urine is sometimes used because it can be recovered in relatively large quantities and is easy to handle (Gaunitz et al. Anal Bioanal Chem. 2019 16 3561-3579. doi: 10.1007 / s00216-019-01837-8).

[0036] The present invention also includes a reagent kit for use in the biomarker detection method of the present invention. The reagent kit of the present invention may include a luciferin precursor and luciferase. A luciferin precursor that reacts specifically with each molecular species of P450 is preferred as the luciferin precursor. Furthermore, it may include a buffer for diluting the sample, etc.

[0037] To aid in understanding the present invention, the present invention will be specifically described below with reference examples and examples, but it goes without saying that the present invention is not limited thereto. In the following examples, KPB refers to 10× potassium phosphate buffer, which is a solution prepared by dissolving 23 g of potassium dihydrogen phosphate (KH2PO4) and 125 g of dipotassium hydrogen phosphate (K2HPO4) in Milli-Q water, making up to 1000 mL, autoclaving, and storing at room temperature until use. Stock Solution Buffer refers to a solution prepared by making up 100 mL of 10× KPB, 200 mL of 100% glycerol, and 2 mL of 0.5 M EDTA (pH 8.0) in Milli-Q water, making up to 1000 mL, autoclaving, and storing at 4 °C until use.

[0038] (Example 1) Confirmation of a biomarker by the P450 luminescence phenomenon in breast cancer In this example, the luminescence phenomenon induced by P450 was observed by reacting luciferin Multi-CYP (Promega, USA), a luciferin precursor, with P450 in the serum of breast cancer patients, and then using the resulting luciferin as a luminescent substrate to react with luciferase and metabolize it. In this example, luciferase produced by genetic recombination was used. Escherichia coli was transformed using a firefly luciferase expression plasmid, which was prepared by inserting firefly-derived luciferase cDNA downstream of the T7 promoter of Biodynamics' expression plasmid pETIA. After culturing the transformed Escherichia coli, the luciferase in the Escherichia coli was affinity-purified using the His tag sequence added to the C-terminus to produce recombinant luciferase. By adding samples collected from test subjects to this reaction system, an attempt was made to detect breast cancer patients based on the change in luminescence values ​​compared to healthy individuals.

[0039] Measurement of the change rate of P450 activity In this example, serum obtained from healthy subjects or breast cancer patients was used as a specimen, and the change rate of P450 activity was measured.

[0040] The collected serum diluted 40-fold with phosphate buffer saline (PBS) was used as a measurement sample, and the control was PBS. The three types of P450 molecular species used in this example were diluted with Stock Solution Buffer so that the measured luminescence value was around 5000. CYP1A1 was diluted 30-fold, CYP1A2 was diluted 40-fold, and CYP1B1 was diluted 50-fold. First, 480 μL of potassium phosphate buffer (KPB), 5×NADPH Regeneration System, and each diluted P450 molecular species with Stock Solution Buffer were mixed in a 1.5 mL tube, and thoroughly pipetted and added to a NUNC 460518 384 well plate22 (white) at 20 μL / well. Subsequently, 16 μL / well of PBS, healthy subject serum diluted 100-fold with PBS, and breast cancer patient serum were added, and incubated at 37°C for 30 minutes. Luciferin-Multi CYP (50 mM) (Promega, USA), a luciferin substrate, was diluted 50-fold to adjust the final concentration to 1 mM and added to the reaction system, and centrifuged with a plate centrifuge, and incubated at 37°C for 40 minutes. In a 1.5 mL tube, 320 μL of luciferase diluted 2-fold with HEPES solution, 25.6 μL of 50 mM ATP, 12.8 μL of MgSO4, 4 μL of inorganic pyrophosphatase, and 277.6 μL of HEPES Triton solution were mixed and added at 40 μL / well, and thoroughly pipetted. Centrifugation was performed with a plate centrifuge, and after standing at room temperature for 10 minutes, the luminescence intensity was measured with a plate reader SH-9000 (Corona Electric Co., Ltd., Japan). P450 was calculated according to the formula shown in Equation 1 below. (3.0 second integrated value)

[0041] The luminescence intensity was measured twice for each specimen, and the measurement was repeated three times. The inhibition rate of the luminescence intensity for each specimen (2 points) was calculated by Equation 2.

[0042] For the specimens described in Table 1, the rates of change in activity were confirmed for three molecular species of CYP1A1, CYP1A2, and CYP1B1 as P450 molecular species, and are shown in FIG. 1. When a t-test was performed on these results, significant differences were confirmed for all of CYP1A1, CYP1A2, and CYP1B1. CYP1A1 HV vs BC (p = 0.00114 < 0.01) There is a significant difference CYP1A2 HV vs BC (p = 0.02522 < 0.05) There is a significant difference CYP1A1 HV vs BC (p = 0.01222 < 0.05) There is a significant difference

[0043] (Example 2) Next, regarding the AUC (Area Under the Roc Curve) values of biomarkers based on the luminescence phenomenon of P450 in breast cancer, the influence of each serum of healthy subjects and breast cancer patients on this luminescence was patterned using a plurality of P450 molecular species by a luminescence competition assay, and a comparison between healthy subjects and breast cancer patients was performed. Based on the results of Example 1, a Receiver Operating Characteristic Curve (ROC curve) was created for each P450 molecular species and is shown in FIG. 2. As a result, AUC = 0.914 for CYP1A1, AUC = 0.790 for CYP1A2, and AUC = 0.852 for CYP1B1. The AUC values obtained from the ROC curve are 0.85 or more for CYP1A1 and CYP1B1, and healthy subjects and breast cancer patients can be accurately discriminated regardless of which P450 molecular species of CYP1A1, CYP1A2, and CYP1B1 is used.

[0044] In this example, only three P450 molecular species were confirmed, but it is considered that by variously examining a plurality of P450 molecular species and luciferin precursors, a more highly accurate breast cancer test biomarker can be developed. From the above results, it was suggested that each molecular species of P450 has the possibility of being used as a biomarker that can accurately distinguish healthy subjects and breast cancer patients.

[0045] (Example 3) Confirmation of a biomarker by P450 luminescence in pancreatic cancer In this example, the luminescence phenomenon due to P450 was observed in the serum of pancreatic cancer patients using the same reaction as in Example 1. By adding samples collected from the subjects to this reaction system, we attempted to detect pancreatic cancer patients based on the change in luminescence values ​​compared to healthy individuals.

[0046] Measurement of P450 activity change rate: In this example, serum obtained from healthy individuals or pancreatic cancer patients was used as a sample, and the P450 activity change rate was measured.

[0047] The collected serum was diluted 50-fold with phosphate-buffered saline (PBS) to be used as the measurement sample, and PBS was used as the control. In this example, the three P450 molecular species used were diluted with Stock Solution Buffer so that the measured luminescence value was around 5000. CYP1A1 was diluted 40-fold, CYP1A2 4-fold, and CYP1B1 50-fold. The measurement principle was carried out according to the description in Example 1.

[0048] For the samples listed in Table 2, the activity change rates for three P450 isoforms, CYP1A1, CYP1A2, and CYP1B1, were examined and are shown in Figure 3. A t-test was performed on these results, and significant changes in the activity rate were observed: p<0.05 for CYP1B1 and p<0.01 for CYP1A1. CYP1A1: Healthy (HV) vs. Pancreatic Cancer (PC) (p<0.01) - Statistical difference. CYP1A2: HV vs. PC; CYP1B1: HV vs. PC (p<0.05) - Statistical difference.

[0049] (Example 4) Next, regarding the AUC value of the biomarker due to the P450 luminescence phenomenon in pancreatic cancer, a comparison was made between healthy individuals and pancreatic cancer patients by patterning the effect of each serum of healthy individuals and pancreatic cancer patients on this luminescence using multiple P450 molecular species through a luminescence competition assay. Based on the results of Example 3, ROC curves were created for each P450 molecular species and are shown in Figure 4. As a result, the AUC was 0.840 for CYP1A1, 0.531 for CYP1A2, and 0.765 for CYP1B1. The AUC values ​​obtained from the ROC curves were 0.75 or higher for CYP1A1 and CYP1B1, indicating that even when using P450 molecular species for CYP1A1 and CYP1B1, healthy individuals and pancreatic cancer patients can be accurately distinguished.

[0050] In this example, only three P450 molecular species were identified. However, by investigating multiple P450 molecular species and luciferin precursors, it is possible to develop more accurate biomarkers for pancreatic cancer detection. The results suggest that each P450 molecular species has the potential to be used as a biomarker that can accurately distinguish between healthy individuals and pancreatic cancer patients.

[0051] (Example 5) Urine samples from depressed patients In this example, the luminescence phenomenon due to P450 was observed in urine samples from depressed patients using the same reaction as in Example 1. By adding urine samples collected from subjects to this reaction system, an attempt was made to detect depressed patients based on the change in luminescence values ​​compared to healthy individuals.

[0052] Measurement of P450 Activity Change Rate: In this example, urine samples were obtained from healthy individuals or patients with depression, and the P450 activity change rate was measured. The activity change rates for three P450 isoforms, CYP1A1, CYP1A2, and CYP1B1, were examined and are shown in Figure 5. When a t-test was performed on these results, p = 0.00486 for CYP1A1, p = 0.015 for CYP1A2, and p = 0.301 for CYP1B1, indicating a significant change in the rate of change for CYP1A1.

[0053] Next, using a luminescence competition assay, the effect of urine from healthy individuals and depressed patients on this luminescence was patterned using multiple P450 molecular species, allowing for a comparison between healthy individuals and depressed patients. Based on the results of the measurement of the P450 activity change rate, ROC curves were created for each P450 molecular species and are shown in Figure 6. As a result, the AUC was 0.844 for CYP1A1 and 0.822 for CYP1A2. The AUC values ​​obtained from the ROC curves were above 0.80 for both CYP1A1 and CYP1A2, indicating that even when using P450 molecular species for CYP1A1 and CYPA2, healthy individuals and depressed patients can be accurately distinguished. In this example, only three types of P450 molecular species were examined, but it is thought that by investigating multiple types of P450 molecular species and luciferin precursors, it may be possible to develop more accurate diagnostic biomarkers for depressed patients. From these results, it is suggested that each P450 molecular species has the potential to be used as a biomarker that can accurately distinguish between healthy individuals and depressed patients.

[0054] The present invention provides a measurement method in which one or more P450s in a sample are reacted with a luminescent substrate precursor, and the luminescence emitted by the metabolism of the luminescent substrate converted from the luminescent substrate precursor is measured. This method makes it possible to easily and accurately detect new biomarkers for diseases in which P450 activity fluctuates. While fluorescence methods require excitation light energy, resulting in background noise during measurement, and require an excitation light source, leading to interference phenomena due to combined fluorescence, the present invention's method for measuring luminescence does not require an excitation light source. Therefore, unlike fluorescence methods which require consideration of background noise during measurement due to excitation light energy, the present invention allows for accurate measurement results even from small amounts of sample, enabling screening of multiple P450s and easy detection of biomarkers related to diseases in which P450 activity fluctuates. Furthermore, for diseases in which one or more P450 activities fluctuate, or are expected to fluctuate, minimally invasive testing with minimal burden on the subject can be performed, allowing for suitable follow-up observation.

Claims

1. A measurement method for testing diseases in which cytochrome P450 activity fluctuates, characterized by reacting one or more types of cytochrome P450 in a sample taken from a subject with a luminescent substrate precursor, and measuring the luminescence emitted when the luminescent substrate converted from the luminescent substrate precursor is metabolized.

2. The measurement method according to claim 1, wherein the disease in which cytochrome P450 activity fluctuates is a disease in which cytochrome P450 activity fluctuates due to inflammation.

3. The measurement method according to claim 1, wherein the luminescent substrate precursor is a luciferin precursor.

4. The measurement method according to claim 1, wherein the luminescent substrate is luciferin, and the luminescence is due to the action of luciferin and luciferase.

5. The measurement method according to claim 1, wherein the cytochrome P450 comprises one or more selected from CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, CYP3A7, CYP4F2, CYP4F3A, CYP4F3B, CYP4F12, and CYP19.

6. The measurement method according to claim 1, wherein the specimen collected from the subject is a minimally invasive specimen.

7. The measurement method according to claim 1, wherein the minimally invasively collected specimen is selected from blood-derived components, urine, saliva, sputum, and sweat.

8. A method for detecting a disease biomarker, comprising reacting one or more types of cytochrome P450 in a sample taken from a subject with a luminescent substrate precursor, and detecting the luminescence pattern of one or more types of cytochrome P450 by measuring the luminescence emitted when the luminescent substrate converted from the luminescent substrate precursor is metabolized.

9. The detection method according to claim 8, wherein the disease is a disease in which the activity of one or more types of cytochrome P450 is expected to fluctuate.

10. An analytical method for testing diseases in which cytochrome P450 activity fluctuates due to inflammation, comprising the following steps: (1) detecting a luminescence pattern in a sample taken from a subject by the measurement method described in any one of claims 1 to 7; (2) analyzing the overlap between the luminescence pattern detected in step (1) and a luminescence pattern previously created from a sample of a disease in which cytochrome P450 activity fluctuates.

11. The analytical method according to claim 10, wherein the disease in which cytochrome P450 activity fluctuates due to inflammation is any disease selected from cancer, neurological diseases, digestive system diseases, respiratory system diseases, and metabolic diseases.

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