KR101824530B1 - Multiplex cytochrome p450 isoenzyme assay - Google Patents

Multiplex cytochrome p450 isoenzyme assay Download PDF

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KR101824530B1
KR101824530B1 KR1020150075295A KR20150075295A KR101824530B1 KR 101824530 B1 KR101824530 B1 KR 101824530B1 KR 1020150075295 A KR1020150075295 A KR 1020150075295A KR 20150075295 A KR20150075295 A KR 20150075295A KR 101824530 B1 KR101824530 B1 KR 101824530B1
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cytochrome
isoenzymes
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isoenzyme
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KR20160139781A (en
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정병화
오현아
이현범
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한국과학기술연구원
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Abstract

The present invention provides a composition for analyzing cytochrome P450 isoenzymatic activity using gas chromatography containing a substrate that specifically reacts with cytochrome P450 isoenzymes to provide various cytochrome P450 isoenzymes Can be measured simultaneously. Accordingly, the present invention can provide a system capable of efficiently measuring the interaction of a drug in a composite containing multi-components such as herbal medicine.

Description

[0002] MULTIPLEX CYTOCHROME P450 ISOENZYME ASSAY [0003]

The present disclosure relates to a method for simultaneously measuring and analyzing the activity of enzymes that function as primary metabolites of a drug in cytochrome P450 isoenzymes using a gas chromatography mass spectrometer.

In recent years, various comparative studies on metabolic enzymes and their activities affecting drug interactions have been emphasized, while encouraging the evaluation of drug interactions using in vitro assays in the development of new drugs. In addition, 82 (about 26%) of the 314 drug-related hepatotoxicity cases reported by the government in 2005 were reported to be due to herbal medicines. However, studies on the drug interactions of herbal medicines combined with various herbal medicines such as toxicity and side effects Is lacking.

Therefore, it is required to evaluate the activity and inhibition method of cytochrome P450 (CYP450), which is mediated by drug interaction and plays a key role in the biotransformation and metabolism of the drug in the liver, .

Experimental protocols for in vitro enzyme induction and inhibition studies are very diverse in each laboratory. In Feb. 2013, the Korea Food & Drug Administration (KFDA) compiled a commentary on the drug metabolism evaluation test method, Does not exist.

According to the recently published Food and Drug Administration's Assessment Method for Drug Metabolism, there are two methods for quantifying the amount of metabolites of Cytochrome P450 enzyme-specific substrate produced from drug-treated hepatocytes. And a method using a liquid chromatography-mass spectrometer.

Fluorescent probes can not be tested on in vtiro , and each enzyme has a disadvantage that is not specific for each isoenzymes. The use of liquid chromatography-mass spectrometry (LC-MS / MS) is more widely used because it involves all of the CYP isoenzymes of the microsomes, so one reaction, one injection, This is because high-throughput screening is possible, and many studies have been made using HPLC or HPLC-UV.

Liquid Chromatography - Mass spectrometry is a pretreatment process to measure enzyme activity. After the enzymatic reaction, proteins are removed. Usually, methanol or acetonitrile is treated, followed by vortexing and centrifugation. The solvent layer is injected into the instrument. However, in order to confirm the activity by treating the complex such as herb medicine with the enzyme, the pretreatment method of protein pretreatment alone can not detect the enzyme product due to the complex, Can not be completely eliminated, and there is a limit to checking the interactions between drugs.

Korean Patent Publication No. 10-2003-0097059 (published on December 31, 2013)

The object of the present invention is to provide a composition capable of efficiently and simultaneously analyzing the activities of various isoenzymes of cytochrome P450, a first-order metabolic enzyme in mammalian drug metabolism. The present invention also provides a method for evaluating the activity of cytochrome P450 isoenzymes using the above composition and a method for evaluating the metabolism of drugs.

According to one aspect of the present invention, there is provided a composition for assaying cytochrome P450 isoenzymatic activity using gas chromatography, which comprises a substrate that specifically binds to cytochrome P450 isoenzymes.

In one aspect of the present invention, the cytochrome P450 isoenzymes are at least one selected from the group consisting of cytochrome P450 1A2, cytochrome P450 2A6, cytochrome P450 2C9, cytochrome P450 2C19, cytochrome P450 CYP2D6, cytochrome P450 CYP2E1 and cytochrome P450 3A4 Wherein the substrate is selected from the group consisting of Phenacetin, Coumarin, Cotinine, Diclofenac, (S) -Mephenytoin, Dextromethorphan, There is provided a composition for assaying cytochrome P450 isoenzymatic activity by gas chromatography comprising at least one member selected from the group consisting of chloroxazone and testosterone.

According to another aspect of the present invention, there is provided a method for assaying cytochrome P450 isoenzymatic activity, comprising the steps of: mixing the cytochrome P450 isoenzyme and a coenzyme to perform an enzyme reaction; Extracting cytochrome P450 isoenzymes from the enzyme reaction product; And quantitating the cytochrome P450 isoenzymes by cytochrome P450 isoenzymes using a gas chromatography mass spectrometer to confirm the activity of cytochrome P450 isoenzymes.

According to another aspect of the present invention, there is provided a method for assaying a cytochrome P450 isoenzymatic activity, comprising the steps of: mixing the isolated cytochrome P450 isoenzymes, Extracting cytochrome P450 isoenzymes from the enzyme reaction product; And analyzing the activity of cytochrome P450 isoenzymes by quantitating the cytochrome P450 isoenzyme product by a gas chromatography mass spectrometer.

The composition according to the present invention includes a substrate that specifically reacts with cytochrome P450 isoenzymes. By gas chromatography mass spectrometry of the composition, mass analysis of the substrate contained in the composition and the products resulting from the enzymatic reaction of the enzyme The activity of various cytochrome P450 isoenzymes can be measured simultaneously.

Since the activity of the cytochrome P450 isoenzymes is analyzed by gas chromatography mass spectrometry, the present invention provides a method of detecting the activity of each enzyme in a complex containing various kinds of cytochrome P450 isoenzymes with superior sensitivity than liquid chromatography mass spectrometry Can be analyzed accurately. ,

Thus, by applying an unknown sample such as a drug to the composition according to the present invention, the metabolism of the unknown sample in the human body can be predicted through the activity analysis of various kinds of cytochrome P450 isoenzymes. Also, according to the assay method of the present invention, activity or inhibition of cytochrome P450 enzyme according to the concentration of an unknown drug sample can be confirmed.

Therefore, according to the present invention, it is possible to provide a system for predicting and preventing the occurrence of side effects of a drug by efficiently measuring the interaction of the drug in a composite containing multiple components such as herbal medicines.

Brief Description of the Drawings Fig. 1 is a diagram showing a main enzyme that functions as a primary metabolite of a drug in human cytochrome P450 isoenzymes.
2 is a chromatogram showing the products of cytochrome P450 isoenzymes using a gas chromatography mass spectrometer.
3A to 3D are graphs showing production rates of products according to the concentration of each substrate of cytochrome P450 isoenzymes.
FIG. 4 is a graph showing the amount of products produced as a result of enzymatic reaction of cytochrome P450 isoenzymes according to reaction time. FIG.
Figures 5A-5D are graphs showing inhibitors of cytochrome P450 isoenzymes treated by concentration.

Hereinafter, embodiments of the present invention will be described in detail.

An embodiment of the present invention provides a composition for assaying cytochrome P450 isoenzymatic activity using gas chromatography, which comprises a substrate that specifically binds to cytochrome P450 isoenzymes.

The cytochrome P450 (CYP, P450) according to an embodiment of the present invention is a heme-protein having a heme as a substituent. When iron ion contained in heme is bonded to carbon monoxide in a reduced state, the maximum absorbance at 450 nm . This enzyme is a typical catalytic enzyme that oxidizes intrinsic substances such as most drugs or environmental substances, endogenous substances such as steroids and carcinogens, and accounts for 80% of primary metabolism of drugs and hormones in living organisms, , Which is a key element in the development of new drugs. The cytochrome P450 enzyme is located in the cell membrane of the endoplasmic reticulum and is contained in the hepatocytes. The microsomal cytochrome P450 system receives electrons from NADPH-cytochrome P450 reductase (NPR) and performs oxidation reaction. As a result, NADPH is oxidized to NADP + , NPR itself is reduced, and P450 and NPR have one electron transport system by oxidizing exogenous substances with the help of reduced P450 enzyme. FIG. 1 shows the main enzymes that perform primary metabolism of drugs in human cytochrome P450 isoenzymes.

The cytochrome P450 isoenzymes according to one embodiment of the present invention are selected from the group consisting of cytochrome P450 1A2 (EC number 1.14.14.1), cytochrome P450 2A6 (EC number 1.14.14.1), cytochrome P450 2C9 (EC numbers 1.14.13.48, 1.14.13.49, 1.14.13.80), cytochrome P450 2C1 (EC numbers 1.14.13.48, 1.14.13.49, 1.14.13.80), cytochrome P450 2D6 (EC number 1.14.14.1), cytochrome P450 2E1 3A4 (EC numbers 1.14.13.157, 1.14.13.32, 1.14.13.67, 1.14.13.97).

In addition, the composition according to the present invention is a substrate that specifically binds to the cytochrome P450 isoenzymes, such as Phenacetin, Coumarin, Cotinine, Diclofenac, (S) -mephenytoin ((S) -Mephenytoin), Dextromethorphan, Chlorzoxazone, and Testosterone.

In one embodiment, the composition according to the present invention is characterized in that the substrate is selected from the group consisting of diclofenac, (S) -mephenytoin and testosterone, a group consisting of phenacetin and coumarin, cotinine, dextromethorphan and chlordoxazone And may include one or more of the above groups. These groups are combined so that products can be effectively generated in consideration of the reaction time depending on the reaction rate of each cytochrome P450 isoenzymes and the K cat (turnover efficiency) in which the product is generated in the substrate.

When multiple substrates are reacted at the same time, there is a difference in reaction rate and reactivity for each substrate, and a large amount of substrate is required to analyze the product with the same sensitivity. Accordingly, the present invention can be performed by dividing the substrates into three groups as described above. For example, among the above substrate groups, the group containing diclofenac, (S) -mephenytoin and testosterone (mixture A) has high extraction efficiency before analysis by GC-MS, and even if a small amount of product is produced after the substrate- It is a grouping of recoverable substrates. The group containing the cotinine, dextromethorphan and chlordoxone (mixture C) had poor extraction efficiency prior to analysis by GC-MS, so many samples should be used to maximize the amount of product produced after the substrate-enzyme reaction Which is a grouping of temperaments. The group containing phenacetin and coumarin (mixture B) is grouped into substrates corresponding to the middle of mixture A and C. As described above, when the substrates are grouped according to the reaction rate and reactivity, the reaction time and the concentration of the biological sample are adjusted when the biological metabolism of the drug sample is analyzed using the unknown biological sample according to the group, , And is effective in maintaining the linearity of the product and preventing potential interactions between the substrates.

According to one more specific embodiment, the cytochrome P450 isoenzymes may be contained in mammalian liver microsomes, primary hepatocytes, cell lines, E. coli, and the like. Drugs and the like are metabolized at the time of administration, and as a more specific example, the cytochrome P450 isoenzymes may be contained in human liver microsomes.

The substrate according to one embodiment of the present invention may be enzymatically reacted with cytochrome P450 isoenzymes to produce a product. The product may be selected from the group consisting of acetaminophen, 7-hydroxycoumarin, 4-Hydroxycycloquinoline, 4-Hydroxydiclofenac, 4-Hydroxymephenytoin, Dextrorphan, 6-Hydroxychlorzoxazone, And 6? -Hydroxytestosterone. The term " a "

In one embodiment, the product may be an extract obtained by extracting a reactant reacted with the cytochrome P450 isoenzymes, and the extract may be a mixture of ethyl acetate and dichloromethane as an extraction solvent.

In the analysis of many types of enzyme substrates and products due to the metabolism of complex drugs such as herbal medicines, when the enzyme reaction solution is directly injected without using the conventional liquid chromatography mass spectrometer and the product is not extracted from the enzyme reaction solution Analytical disturbance peaks are generated and precise quantification is difficult. Thus, there is a further need for an extraction process to remove analytical interfering materials. However, in one embodiment, the present invention uses gas chromatography mass spectrometry for the activity analysis of the cytochrome P450 isoenzymes to perform various processes required for analysis such as preparation of mobile phase or column conditioning, unlike liquid chromatography mass spectrometry There is no need and the resolution of the analysis is good, so it is easy to repeatedly analyze the sample repeatedly. In one embodiment, the present invention can measure the activity of cytochrome P450 isoenzymes by gas chromatography mass spectrometry of the composition for cytochrome P450 isoenzymatic activity assay described above.

Specifically, one embodiment of the present invention comprises the steps of: preparing a composition for assaying cytochrome P450 isoenzymes, mixing the cytochrome P450 isoenzymes and a coenzyme, and performing an enzyme reaction; Extracting cytochrome P450 isoenzymes from the enzyme reaction product; And analyzing the activity of the cytochrome P450 isoenzymes by quantitating the cytochrome P450 isoenzymes by mass spectrometry using a gas chromatograph mass spectrometer.

Furthermore, one embodiment of the present invention can analyze the metabolism of an unknown sample, such as a drug, by applying the above-described cytochrome P450 isoenzyme activity assay method, and confirm the drug interaction in the human body.

As a specific example, the present invention relates to a method for preparing a cytochrome P450 isoenzymatic activity analyzing composition comprising the above-described composition for cytochrome P450 isoenzymatic activity assay, an isolated biological sample containing the cytochrome P450 isoenzymes, a coenzyme and a drug, Extracting cytochrome P450 isoenzymes from the enzyme reaction product; And analyzing the activity of cytochrome P450 isoenzymes by quantitating the cytochrome P450 isoenzyme product with a gas chromatography mass spectrometer.

In one embodiment, the step of extracting the cytochrome P450 isoenzyme product may include treating the enzyme reaction product with a mixture of ethyl acetate and dichloromethane as an extraction solvent to extract cytochrome P450 isoenzyme product. Use of the above mixture can effectively remove the interfering substances in the enzyme activity assay of the present invention, thereby improving the extraction efficiency. As an example, the product may be an extract obtained by basically treating the reaction between the cytochrome P450 isoenzymes and a substrate, and extracting a mixture of ethyl acetate and dichloromethane as an extraction solvent.

The mixture may contain ethyl acetate in an amount of at least 1 vol%, at least 10 vol%, at least 20 vol%, at least 30 vol%, at least 40 vol%, at least 50 vol%, at least 60 vol%, at least 70 vol% , Not less than 80 vol%, or not less than 90 vol%, and not more than 99 vol%, not more than 90 vol%, not more than 80 vol%, not more than 70 vol%, not more than 60 vol%, not more than 50 vol%, not more than 40 vol% , Not more than 30% by volume, not more than 20% by volume, or not more than 10% by volume, but is not limited to the above range as long as the reaction product can be effectively extracted. The mixture may contain up to 99 vol%, up to 90 vol%, up to 80 vol%, up to 70 vol%, up to 60 vol%, up to 50 vol%, up to 40 vol%, up to up to 30 vol% , 20 vol% or less, 10 vol% or less, and 1 vol% or more, 10 vol% or more, 20 vol% or more, 30 vol% or more, 40 vol% or more, 50 vol% or more, 60 vol% or more , Not less than 70% by volume, not less than 80% by volume, or not less than 90% by volume as long as the reaction product can be effectively extracted. In a more specific embodiment, the mixture may be a mixture of ethyl acetate and dichloromethane in a volume of 20-80: 80-20, 30-70: 70-30 or 40-60: 60-40.

The coenzyme is exemplified by NADP + and the like, and is not limited thereto as long as it helps the reaction of the cytochrome P450 isoenzymes with the substrate. In one embodiment, a metal ion such as MgCl 2 and a chelate ion such as ethylenediamine-tetraacetic acid (EDTA) may be further added to the auxiliary agent, and the auxiliary agent may temporarily bind to the enzyme The enzyme acts as a reducing enzyme. The chelate ion may aid lipid peroxidation in the presence of microsomes and NADPH.

In one embodiment, the activity of the cytochrome P450 isoenzymes is analyzed by mass spectrometry using MSTFA (N-methyl-N- (trimethylsilyl) -trifluoroacetamide), NH 4 I and DTE (dithioerythritol) may be added and heat treatment may be performed to further derivatize the cytochrome P450 isoenzymes. More specifically, in the derivatization step, the nitrogen is completely removed from the extracted cytochrome P450 isoenzymatic product, and then stored in a desiccator in a vacuum state for at least 30 minutes. Then, MSTFA: NH 4 I: DTE (500: 4: 2 = w: v: v), and treating in a 60 heat block for 30 minutes. When the enzyme is derivatized as described above, the cytochrome P450 isoenzymes and the substrates can raise the recovery rate of the product produced through the enzymatic reaction to nearly 100%, and the problem of the sensitivity of the product which may be caused by gas chromatography mass spectrometry Can be solved.

In one embodiment, the step of analyzing the activity of the cytochrome P450 isoenzymes may further comprise determining that the activity of the cytochrome P450 isoenzymes is high if the concentration of cytochrome P450 isoenzymes is high as determined by gas chromatography mass spectrometry have. As a more specific example, the enzyme activity may be expressed as the concentration of the product relative to the concentration of the substrate specifically binding to the cytochrome P450 isoenzymes, wherein the substrate reacts with the enzyme and cytochrome P450 isoenzymes, And the enzyme activity is higher as the concentration of the product increases. In view of the above, an embodiment of the method for evaluating bio-metabolism of a drug may further include a step of evaluating the activity of the cytochrome P450 isoenzymes as a result of analyzing the activity of the cytochrome P450 isoenzymes to be high if the metabolism of the drug is high.

The biological sample may be, for example, a liver microsome of a mammal, more specifically, a human liver microsome, but is not limited as long as it can confirm the metabolism of a drug. In one embodiment, the drug may be a mixture of one or more drugs, and more specifically, it may be a herbal medicine containing a plurality of drugs.

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to examples. However, the following embodiments are provided for illustrative purposes only for the sake of understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

[Example 1] Production of standard solution of analytes and gas chromatography analysis

1-1. Cytochrome P450 Isozyme  decision

In the present invention, to determine the activity of cytochrome P450, seven substrates and products of isoenzymes that can be detected by gas chromatography mass spectrometry (GC-MS) were determined.

P450 isoenzymes temperament product CYP1A2 Phenacetin Acetaminophen CYP2A6 Coumarin 7-Hydroxycoumarin < / RTI > Cotinine 3-Hydroxycotinine < RTI ID = 0.0 > CYP2C9 Diclofenac 4-Hydroxydiclofenac < / RTI > CYP2C19 (S) -mephenytoin ((S) -Mephenytoin) 4-Hydroxymephenytoin < / RTI > CYP2D6 Dextromethorphan Dextrorphan CYP2E1 Chlorzoxazone 6-Hydroxychlorzoxazone < / RTI > CYP3A4 Testosterone 6β-Hydroxytestosterone (6β-Hydroxytestosterone)

Two cytochrome P450 isoenzymes, CYP2A6 and coumarin, were used as substrates. This is because coumarin is frequently detected as a component of herbal medicines. When analyzing herbal medicine, if coumarin alone is used as a substrate, the drug interaction of the cytochrome P450 enzyme can not be known due to the coumarin already present in the herbal medicine herb. The activity of the enzyme by the enzyme can not be predicted.

The products resulting from the enzymatic reactions of each cytochrome P450 isoenzymes listed in Table 1 above were purchased from Sigma Aldrich, Santa Cruz Technology and BD Science to determine the gas chromatographic conditions. The stock solution was diluted to 50 μg / mL with 1000 μg / mL of methanol. Carbamazepine was used as an internal reference material.

1-2. Gas Chromatography-Mass Spectrometer ( GC -MS) method

The products of each cytochrome P450 isoenzymes were analyzed using gas chromatography-mass spectrometry (GC-MS). Gas chromatography and mass spectrometry were performed on an Agilent Technologies 7890A GC system and a 5975C VL MSD model, and the columns were analyzed with DB-5MS (30m × 0.25 mm, 0.25 μm). The oven temperature was sampled at 280 in an aliquot manner to a ratio of 12 to 1, and the sample injection volume was 1 μL and the flow rate was 1 mL / min. The temperature of the oven was set at a temperature of 20 / min with an initial temperature of 100, and the temperature was controlled by keeping the temperature from 190 to 10 / min to 300 for 1 minute. The helium gas was used as the mobile phase to stabilize physically and chemically, and the selective ionization monitoring method was used. The source temperature of the mass spectrometer was 230, the ionization voltage was 70 eV, and the solvent delay time was 5 minutes.

[Example 2] Extraction method and reaction conditions of cytochrome P450

2-1. Reaction conditions of cytochrome P450 enzyme

The human liver microsome required for the cytochrome P450 enzyme reaction was purchased from BD Science (Q452117) and used at a concentration of 20 mg / mL. NADPH and MgCl 2 were also purchased from Sigma-Aldrich as coenzymes to aid the reaction. The isoenzymatic substrate and the human-liver microsomal concentration used in the experiment were determined according to the K m value by selecting the optimum reaction conditions suitable for GC-MS analysis. In addition, the reaction time was optimized according to the reaction rate of each isoenzymes. The mixture A was divided into Diclofenac, (S) -mephenytoin and Tasstosterone, B was phenacetin and coumarin, C was cotinine with other substrates, dextro metro plate And chlorosuccinate. Details of the substrate mixtures A, B, and C are listed in Table 2 below.

Classification temperament Substrate Concentration (μM) Microsomal concentration (mg / mL) Mixture A Diclofenac 20 0.25 (S) -mephenytoin 20 Testosterone 150 Mixture B Phenacetin 150 0.5 Coumarin 5 Mixture C
Cotinine 100 One Dextrose 100 Chlorous 100

Mixture A was mixed with 1.25 μL and 8.75 μL of human liver microsomes (concentration 20 mg / mL) and 0.1 M potassium phosphate buffer (potassuim phasphate buffer), respectively. Mixture B contained human liver microsomes (concentration 20 mg / mL) 2.5 μL and 7.5 μL of 0.1 M potassium phosphate buffer and 5 μL of human hepatic microsomes (concentration 20 mg / mL) and 0.1 M potassium phosphate buffer (pH 5), respectively. Lt; / RTI > The final concentrations of each mixture A, B, and C were optimized to 0.23 to 0.28 mg / mL, 0.48 to 0.52 mg / mL, and 0.98 to 1.02 mg / mL. The reaction components except for NADPH were mixed and pre-incubated for 5 minutes in a 37 shaking incubator, and then the reaction was initiated by adding 20 μL of NADPH (concentration 50 mM). After reacting for 15 minutes, the reaction was stopped by treating with 197 μL of cold ammonium bicarbonate (NH 4 HCO 3 ) of pH 9 and immediately stored in ice. All reactions were repeated 3 times and the detailed capacity of the reactions was posted in Table 3 below.

Reaction conditions Volume Mixture A Mixture B Mixture C Substrate (μL) One One One Person - liver microsome
(μL, 20 mg / mL) 1.25 2.5 5 0.1 M Potassium phosphate Buffer capacity (μL) 8.75 7.5 5 NADPH (μL, 50 mM) 20 20 20 MgCl 2 (μL, 100 mM) 5 5 5 Tertiary distilled water (μL) 64 64 64 Total Reaction Capacity (μL) 100 100 100 Ammonium bicarbonate buffer
Capacity (μL) 197 197 197 Pre-incubation time (min) 5 5 5 Reaction time (min) 15 15 50

2-2. Cytochrome P450  Extraction of enzyme products

After each enzymatic reaction of the cytochrome P450 isoenzymes and the substrate was terminated, 3 μL (1000 μg / mL) of carbamazepine, an internal standard, was added. For the first extraction, 1 mL of ethyl acetate was added, vortexed for 1 minute, and centrifuged at 4, 3500 rpm for 4 minutes. After transferring the supernatant from the separated layer to a new test tube, 1 mL of dichloromethane was added to the second extraction, followed by vortexing and centrifugation. The organic solvent layer was completely blown with nitrogen along with the supernatant of the first extraction. This was stored in a desiccator until it was derivatized. The ratio of the final ethyl acetate to dichloromethane was 1: 1 (v: v).

2-3. Cytochrome P450 according to extraction solvent Isozyme  Confirm extraction efficiency of product

In order to confirm the extraction efficiency of cytochrome P450 isoenzymes according to the various extraction methods as described in 2.2 above, firstly, ethyl acetate and dichloromethane treated with pH of the stop solvent of 3 and 9, Solvent, respectively. Finally, the acetonitrile protein treatment was carried out to confirm the extraction efficiency. All experiments were repeated 3 times, and the results are listed in Table 4.

product Basic stop-ethyl acetate-dichloromethane Acid stop-
Ethyl acetate-dichloromethane Ethyl acetate Dichloromethane Acetonitrile Acetaminophen 89.1 32.6 70.5 23.7 65.4 7-Hydroxycoumarin < / RTI > 97.5 74.9 82.1 108.1 65.8 3-Hydroxycotinine < RTI ID = 0.0 > 101.4 8.0 14.6 63.0 60.6 4-Hydroxydiclofenac < / RTI > 88.5 74.8 80.5 24.7 68.2 4-Hydroxymephenytoin < / RTI > 95.7 71.0 70.1 83.7 72.6 Dextrorphan 84.8 42.0 26.1 65.9 85.0 6-Hydroxychlorzoxazone < / RTI > 115.8 67.1 82.1 98.1 124.3 6β-Hydroxytestosterone (6β-Hydroxytestosterone) 121.4 68.6 94.1 100.4 57.0

As shown in Table 4, when the enzyme reaction was basically terminated and the reaction mixture was extracted with a mixture of ethyl acetate and dichloromethane, the yields of all the products of the eight cytochrome P450 isoenzymes were close to 100%. Therefore, in the experiment described below, a product obtained by extracting the basic stop-ethyl acetate-dichloromethane as an extraction solvent was used.

2-4. Of cytochrome P450 enzyme Derivatization

NH 4 I: DTE (dithioerythritol) (500: 4) was added to the MSTFA (N-methyl-N- (trimethylsilyl) : 2 = w: v: v) was added and treated for 30 min at 60 heat block. After cooling, it was transferred to a dedicated vial, and then 1 μL was injected into the gas chromatography.

[Example 3] Identification of activity of cytochrome P450 isoenzymes

1-2. After the gas chromatogram conditions of the products of each cytochrome P450 isoenzymes were determined as described in the gas chromatography-mass spectrometry (GC-MS) analysis, the enzymatic reaction was carried out in vitro and analyzed using a gas chromatography-mass spectrometer Enzyme activity was confirmed.

3-1. Cytochrome P450 Isozyme  Determine the chromatogram of the product

The chromatogram results of each cytochrome P450 isoenzyme product are shown in FIG. The information of the identified chromatogram is shown in Table 5.

P450 isoenzymes product Identified m / z Adduct Retention time (minutes) CYP1A2 Acetaminophen 181 [MC 3 H 6 OSi + TMS + H] + 7.96 CYP2A6 7-Hydroxycoumarin < / RTI > 219 [M + TMS-H] < + > 8.74 3-Hydroxycotinine < RTI ID = 0.0 > 249 [M + TMS-CH 3] + 9.22 CYP2C9 4-Hydroxydiclofenac < / RTI > 302 [M + TMS-CO 2 -HCl] + 14.85 CYP2C19 4-Hydroxymephenytoin < / RTI > 349 [M + 2TMS-2CH 3] + 11.85 CYP2D6 Dextrorphan 329 [M + TMS-H] < + > 12.10 CYP2E1 6-Hydroxychlorzoxazone < / RTI > 286 [M + 2TMS-3CH 3 + 3H] + 10.07 CYP3A4 6β-Hydroxytestosterone (6β-Hydroxytestosterone) 520 [M + 2 < / RTI > TMS-2H] + 16.70 IS Carbamazepine 193 [M + H] < + > 13.01

The carbamazepine in Table 5 is an internal standard, which is intended to correct errors in extraction during analysis.

As shown in FIG. 2, according to the analysis method according to one embodiment of the present invention, the concentration of 8 kinds of stochrome P450 isoenzyme products was measured at the same time, and thus it is possible to simultaneously analyze various kinds of stochrome P450 isoenzymatic activities can confirm.

3-2. Cytochrome P450 Isozyme  Identify the rate of product formation

To determine the reaction rate of each cytochrome P450 isoenzymes, the 8 substrates were treated at 0, 1, 2, 5, 10, 20 and 40 mM concentrations (final substrate concentrations were 0, 10, 20, 50, 100, 200, 400 μM, cotinine 0, 20, 50, 100, 500 μM). As a result, one-half of the concentration K m value for the rate at which the saturation as the unit increasing the concentration of K cat and the substrate can move in rotation metabolic product from the substrate per hour and efficiency of the enzyme velocity by dividing these constants (K cat / Km ) are shown in Figs. 3a to 3d and Table 6.

P450 isoenzymes temperament K cat (min -1 ) K m (μM) K cat / K m M-1 min -1 CYP1A2 Phenacetin 1.19 156.6 0.007 CYP2A6 Coumarin 2.23 7.19 0.310 Cotinine 0.03 54.5 0.001 CYP2C9 Diclofenac 9.60 2.09 0.442 CYP2C19 (S) -mephenytoin ((S) -Mephenytoin) 0.10 31.6 0.003 CYP2D6 Dextromethorphan 0.07 7.4 0.009 CYP2E1 Chlorzoxazone 0.02 296.4 0.001 CYP3A4 Testosterone 13.86 162.2 0.085

As shown in Table 6, coumarin, diclofenac, and dextromethorphan exhibited a very low K m value compared to other substrates, indicating that the enzyme rapidly converts the product to the substrate. That is, it means that the binding affinity of the enzyme to the substrate is high. The other substrates, on the contrary, showed a high K m value, indicating that the binding affinity for the enzyme substrate is low.

3-3. Each cytochrome P450 Isozyme Amount of product  compare

The amounts of products of each cytochrome P450 isoenzymes were compared according to reaction time. The reaction was carried out for 0, 5, 10, 20, 40, 60, 80 min. The amount of product reacted for 80 min was defined as 100%, and the products were compared at each time. This was done three times each, and the reaction results are posted in FIG.

From FIG. 4, it can be seen that the production rate of 7-hydroxycoumarin, 4-hydroxydiclofenac, and dextro-phan decreases with time as the reaction time approaches 100%. This means that the substrate is changed by the enzyme to the product, that is, the substrate-product conversion is fast.

[Example 4] Confirmation of inhibition of Cytochrome P450 isoenzymes

The inhibitory effect of each of the above enzymes was confirmed by treating the cytochrome P450 isoenzymes with concentrations of inhibitors known in the art (the final inhibitor concentrations were 0, 0.1, 0.2, 0.5, 1, 2, 5, 10 μM). The IC 50 value, which is the inhibitory inhibitor concentration of 50% of the enzyme, was determined using this. Table 7 shows the IC 50 values for the substances known as inhibitors of each cytochrome P450 isoenzymes and their concentrations. 5A to 5D show graphs in which the inhibitor is treated for each concentration.

P450 isoenzymes temperament Inhibitor IC 50 ([mu] M) CYP1A2 Phenacetin alpha -naphthoflavone < / RTI > 0.032 CYP2A6 Coumarin 8-Methoxypsoralen < / RTI > 0.488 Cotinine 0.409 CYP2C9 Diclofenac Sulfaphenazole 1.943 CYP2C19 (S) -mephenytoin ((S) -Mephenytoin) S-Benzylnirvanol < / RTI > 0.127 CYP2D6 Dextromethorphan Qunidine 0.124 CYP2E1 Chlorzoxazone Diethyldithiocarbamate < RTI ID = 0.0 > 3.306 CYP3A4 Testosterone Ketoconazole 0.160

Thus, according to one embodiment of the present invention, the activity of cytochrome P450 isoenzymes was measured by gas chromatography, and the inhibitory effect of each well-known cytochrome P450 isoenzymes was treated at different concentrations, Can be confirmed. Therefore, it means that the substrate and the unknown sample according to the present invention can be treated with cytochrome P450 isozyme-containing microsomes to activate or inhibit the cytochrome P450 enzyme of an unknown sample according to the concentration.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (19)

delete delete delete delete delete delete A composition for analyzing cytochrome P450 isoenzymatic activity comprising a substrate that specifically binds to cytochrome P450 isoenzymes; a step of mixing the cytochrome P450 isoenzymes and a coenzyme to perform an enzyme reaction;
Extracting cytochrome P450 isoenzymes from the enzyme reaction product; And
Determining the activity of cytochrome P450 isoenzymes by quantitating the cytochrome P450 isoenzyme product with a gas chromatograph mass spectrometer;
Lt; RTI ID = 0.0 > P450 < / RTI > isoenzyme activity assay. [Claim 7] The method according to claim 7, wherein the activity of the cytochrome P450 isoenzymes is analyzed by gas chromatography mass spectrometry. When the cytochrome P450 isoenzyme product concentration is higher than that of the cytochrome P450 isoenzyme activity assay, And determining that the activity of the enzyme is high. 8. The method of claim 7, wherein the step of extracting the cytochrome P450 isoenzymatic product comprises treating cytochrome P450 isoenzymes with a mixture of ethyl acetate and dichloromethane as an extraction solvent to extract cytochrome P450 isoenzymes Activity analysis method. The method according to claim 7, wherein the activity of the cytochrome P450 isoenzymes is analyzed by using MSTFA (N-methyl-N- (trimethylsilyl) -trifluoroacetamide) NH 4 I and DTE (dithioerythritol), followed by heat treatment to further derivatize the cytochrome P450 isoenzymes. A composition for analyzing the activity of cytochrome P450 isoenzymes comprising a substrate that specifically binds to cytochrome P450 isoenzymes, a step of enzymatic reaction by mixing a separated biological sample containing the cytochrome P450 isoenzymes, a coenzyme and a drug, ;
Extracting cytochrome P450 isoenzymes from the enzyme reaction product; And
Analyzing the cytochrome P450 isoenzymatic activity of the cytochrome P450 isoenzymes by quantitating the cytochrome P450 isoenzyme product with a gas chromatography mass spectrometer;
Wherein the method comprises the steps of: 12. The method according to claim 11, wherein the activity of the cytochrome P450 isoenzymes is analyzed to determine that the cytochrome P450 isoenzymatic activity is higher than that of the composition for analyzing the cytochrome P450 isoenzyme activity, ≪ / RTI > 12. The method according to claim 11, wherein the biological sample is liver microsomes of a mammal. 12. The method according to claim 11, wherein the drug is a mixture of two or more drugs. 8. The method of claim 7,
Wherein the cytochrome P450 isoenzymes include at least one selected from the group consisting of cytochrome P450 1A2, cytochrome P450 2A6, cytochrome P450 2C9, cytochrome P450 2C19, cytochrome P450 CYP2D6, cytochrome P450 CYP2E1 and cytochrome P450 3A4,
The substrate may be selected from the group consisting of Phenacetin, Coumarin, Cotinine, Diclofenac, (S) -mephenytoin (S) -Mephenytoin, Dextromethorphan, Chlorzoxazone) and testosterone (Testosterone).
Cytochrome P450 isoenzyme activity assay. 8. The method of claim 7,
Diclofenac, (S) -mephenytoin and testosterone;
A group consisting of phenacetin and coumarin; And
A group consisting of cotinine, dextromethorphan and chlordoxazone;
Lt; RTI ID = 0.0 > P450 < / RTI > isoenzyme activity. 12. The method of claim 11,
Wherein the cytochrome P450 isoenzymes include at least one selected from the group consisting of cytochrome P450 1A2, cytochrome P450 2A6, cytochrome P450 2C9, cytochrome P450 2C19, cytochrome P450 CYP2D6, cytochrome P450 CYP2E1 and cytochrome P450 3A4,
The substrate may be selected from the group consisting of Phenacetin, Coumarin, Cotinine, Diclofenac, (S) -mephenytoin (S) -Mephenytoin, Dextromethorphan, Chlorzoxazone) and testosterone (Testosterone).
METHODS FOR EVALUATING BODY METABOLISM OF DRUGS 12. The method of claim 11,
Diclofenac, (S) -mephenytoin and testosterone;
A group consisting of phenacetin and coumarin; And
A group consisting of cotinine, dextromethorphan and chlordoxazone;
Or a pharmaceutically acceptable salt thereof. 12. The method of claim 11, wherein the step of extracting the cytochrome P450 isoenzymatic product comprises treating a mixture of ethyl acetate and dichloromethane as an extraction solvent with the enzyme reactant to extract a cytochrome P450 isoenzymatic product, Evaluation method of action.
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