Metabolite for identifying tryptamine new psychoactive substance 4-HO-DET human liver microsome model and application of metabolite

Abstract

The invention discloses a metabolite for identifying a tryptamine new psychoactive substance 4-HO-DET human liver microsome model and application of the metabolite, and relates to the technical field of liver microsomes. The situation that new psychoactive substances (NPS) are abused in a large amount due to uncontrolled conditions in the global range causes serious social, medical and environmental problems. The tryptamine substance 4-HO-DET, as a large class of new psychoactive substances, is rapidly metabolized after being ingested by a human body, and the concentration of an original drug reserved in biological samples such as hair, urine and blood of the human body is extremely low, so that certain difficulty is brought to detection and monitoring of the tryptamine new psychoactive substances. At present, metabolism detection of tryptamine new psychoactive substances is less at home and abroad, and only a small part of tryptamine substances are controlled. Therefore, research on the metabolic process of the tryptamine substances, analysis of the metabolites and metabolic pathways of the tryptamine substances and determination of the metabolic markers of the tryptamine substances play an important role in drug banning work. A human liver microparticle in-vitro model is established for 4-HO-DET, and the metabolic process is simulated.

Description

Technical Field

[0001] The present invention relates to the technical field of liver microsomes, in particular to metabolites identified by a human liver microsome model of a new tryptamine psychoactive substance 4-HO-DET and applications thereof. Background Art

[0002] 4-HO-DET (4-hydroxy-2,3-dihydro-1,4-benzodiazepin-2-one) is a compound with potential pharmacological activity. In the field of drug development, in-depth research on compounds with specific pharmacological activities often reveals their potential applications in treating certain diseases. As a novel compound, the exploration and study of the pharmacological activity of 4-HO-DET is of great significance for promoting new drug development.

[0003] Liver microsomes play a crucial role in drug metabolism. Liver microsomes, organelles within hepatocytes, contain a variety of drug-metabolizing enzymes that catalyze oxidation, reduction, and hydrolysis of various drugs, thereby inactivating them or generating new active metabolites. Therefore, understanding the metabolic characteristics of 4-HO-DET in liver microsomes is crucial for evaluating its pharmacological activity, predicting drug interactions, and guiding new drug development.

[0004] Although 4-HO-DET has attracted the attention of researchers as a compound with potential pharmacological activity, the existing technologies for studying the metabolic characteristics of 4-HO-DET in liver microsomes and its metabolites still have the following shortcomings:

[0005] 1. Unclear metabolic properties: Currently, there is a lack of systematic research on the metabolic properties of 4-HO-DET in liver microsomes. For example, key questions remain, such as which drug-metabolizing enzymes are involved in the metabolism of 4-HO-DET and how these enzymes affect the metabolic rate and metabolites of 4-HO-DET.

[0006] 2. Unknown metabolites: Due to a lack of understanding of the metabolic properties of 4-HO-DET in liver microsomes, its metabolites are also unknown. This poses great difficulties in evaluating the pharmacological activity of 4-HO-DET, predicting drug interactions, and guiding new drug development.

[0007] 3. Limited research methods: Current research on the metabolism of 4-HO-DET in liver microsomes relies primarily on in vitro experimental methods, such as liver microsome incubation experiments. However, these methods often provide only limited metabolic information and fail to fully simulate the true metabolic environment in the human body. Therefore, more comprehensive and accurate research methods are needed to gain a deeper understanding of the metabolic characteristics of 4-HO-DET and its metabolites in liver microsomes. Summary of the Invention

[0008] The present invention provides a metabolite of a tryptamine-type new psychoactive substance 4-HO-DET identified by a human liver microsome model and its application, in order to solve the problems in the background technology.

[0009] To achieve the above objectives, the present invention provides the following technical solutions: metabolites identified by a human liver microsome model of a new tryptamine psychoactive substance 4-HO-DET and their applications, comprising establishing an in vitro human liver microsome incubation model, incubating 4-HO-DET with mixed human liver microsomes at 37° C., adding an NADPH regeneration system to the incubation system to initiate the in vitro metabolic reaction, selecting an incubation time of 90 minutes to ensure that abundant metabolites are obtained, and analyzing the metabolites using ultra-performance liquid chromatography Q-Exactive tandem quadrupole-electrostatic field orbitrap high-resolution mass spectrometry. Data are collected in Full MS-ddMS2 mode, and the structures of the metabolites are deduced based on the mass-to-charge ratio information of the original drug and fragment ions. At least 7 metabolites are identified, including 6 phase I metabolites and 1 phase II metabolite, and the main metabolic pathways are hydroxylation reaction and conjugation reaction with glucuronic acid after hydroxylation.

[0010] Furthermore, the concentration of the mixed human liver microsomes is 20 mg / ml.

[0011] Furthermore, the NADPH regeneration system includes NADP+, G-6-P, G-6-PDH, MgCl2 and UDPGA.

[0012] Furthermore, the final volume of the incubation system is 200 μl, which contains 1.3 mmol / l NADP+, 3.3 mmol / l G-6-P, 0.4 U / ml G-6-PDH, 3.3 mmol / l Mg 2+ , 2.5mmol / L UDPGA.

[0013] Furthermore, in step (d), the chromatographic column used was Waters UPLC HSS T3 (150×2.1 mm, 1.8 μm), the mobile phase A was 0.1% formic acid in water, B was 0.1% formic acid in acetonitrile, the gradient elution program was 0-1 min: 1% B, 1-8 min: 1% A-99% B, 8-10 min: maintain 99% B, 10-12 min: 1% B, and the flow rate was 0.3 mL / min.

[0014] Furthermore, in step (d), the mass spectrometry conditions adopted a heatable electrospray ionization source (H-ESI), positive ion mode acquisition, collision gas was nitrogen (N2), the spray voltage was 3800 V, the vaporization temperature was 320°C, the nebulizer gas pressure was 38 arb, the auxiliary gas pressure was 15 arb, the capillary temperature was 350°C, the full scan acquisition data resolution was 35000, and the secondary mass spectrometry acquisition data resolution was 17500.

[0015] Furthermore, the phase I metabolites include hydroxylated metabolites, dihydroxylated metabolites, carboxylated metabolites, ketone-forming metabolites and dealkylated metabolites.

[0016] Furthermore, the phase II metabolite is a product of a hydroxylation reaction with glucuronic acid.

[0017] Furthermore, the method also includes processing and analyzing the mass spectrometry data by Mass Frontier software and Compound Discover software to deduce the structure of the metabolites and identify unknown metabolites.

[0018] Furthermore, this method can be used for metabolic detection, metabolic pathway analysis, and determination of metabolic markers in drug control work.

[0019] Compared with the prior art, the present invention provides metabolites of tryptamine-type new psychoactive substances 4-HO-DET identified by human liver microsome model and their applications, which have the following beneficial effects:

[0020] 1. Metabolites of the tryptamine-type new psychoactive substance 4-HO-DET were identified in the human liver microsome model and their applications. The metabolic stability of 4-HO-DET in liver microsomes was evaluated, which helps to understand the transformation and elimination process of drugs in the body and provides important information for drug pharmacokinetic studies.

[0021] 2. Metabolites identified by the human liver microsome model of 4-HO-DET, a new tryptamine psychoactive substance, and their applications. By studying the metabolic pathways and metabolites of 4-HO-DET in liver microsomes, the possible efficacy and toxicity of drugs can be predicted, providing a basis for the safety and efficacy evaluation of drugs.

[0022] 3. Metabolites of the tryptamine-type new psychoactive substance 4-HO-DET were identified using the human liver microsomal model and their applications. The inhibitory effect of 4-HO-DET on liver microsomal enzymes was evaluated, which helps to understand the impact of drugs on drug-metabolizing enzymes in the body and thus predict the risk of interaction between drugs and other drugs.

[0023] 4. The metabolites identified by the human liver microsomal model of the new tryptamine psychoactive substance 4-HO-DET and their applications, by studying the inhibition type and inhibition intensity of 4-HO-DET on liver microsomal enzymes, can provide important reference for clinical drug guidance and avoid adverse reactions caused by drug interactions.

[0024] 5. The metabolites identified by the human liver microsome model of the new tryptamine psychoactive substance 4-HO-DET and their applications. The research results of the 4-HO-DET liver microsome briefing can provide an important reference for drug research and development, helping researchers to understand the metabolic characteristics and enzyme inhibition properties of drugs in the body, thereby optimizing drug structure and improving the metabolic stability and safety of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The chemical structure of the tryptamine-type new psychoactive substance 4-HO-DET of the present invention;

[0026] Figure 2 It is the experimental reagent of the present invention;

[0027] Figure 3 The apparatus and equipment of the present invention;

[0028] Figure 4 It is the gradient elution procedure of the present invention;

[0029] Figure 5 The figure is a bar graph showing the relationship between the peak area of ​​4-HO-DET metabolites and the incubation time of the present invention;

[0030] Figure 6 The in vitro metabolite information of the invented 4-HO-DET is provided. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] See also Figure 1-6The invention discloses metabolites identified by the human liver microsome model of 4-HO-DET, a new psychoactive substance of the tryptamine class, and their applications, including establishing an in vitro incubation model of human liver microsomes, incubating 4-HO-DET with mixed human liver microsomes at 37°C, adding an NADPH regeneration system to the incubation system to initiate the in vitro metabolic reaction, selecting an incubation time of 90 minutes to ensure the acquisition of abundant metabolites, and analyzing the metabolites using ultra-performance liquid chromatography Q-Exactive tandem quadrupole-electrostatic field orbital trap high-resolution mass spectrometry. Data is collected in Full MS-ddMS2 mode, and the structure of the metabolites is deduced based on the mass-to-charge ratio information of the original drug and fragment ions. At least 7 metabolites are identified, including 6 phase I metabolites and 1 phase II metabolite. The main metabolic pathways are hydroxylation reaction and hydroxylation followed by conjugation with glucuronic acid.

[0033] Specifically, the concentration of the mixed human liver microsomes is 20 mg / ml.

[0034] In this embodiment, the liver microsomal enzyme system is a complex enzyme system, including phase I metabolic enzymes (such as cytochrome P450, flavin monooxygenase, monoamine oxidase, etc.) and phase II metabolic enzymes (such as glucuronyl transferase, sulfotransferase, etc.). These enzymes play a vital role in drug metabolism and biotransformation. At a concentration of 20 mg / ml, the enzyme content in liver microsomes is high, which is conducive to accurately detecting enzyme activity in in vitro metabolic experiments, thereby more accurately predicting drug metabolism in the human body.

[0035] Specifically, the NADPH regeneration system includes NADP+, G-6-P, G-6-PDH, MgCl2 and UDPGA.

[0036] In this embodiment, NADPH is an important coenzyme for various metabolic reactions in the body, particularly in redox reactions. In drug metabolism, many Phase I reactions (such as hydroxylation and oxidation) require NADPH as a reducing agent. The NADPH regeneration system can continuously provide NADPH to support the progress of these metabolic reactions.

[0037] Specifically, the final volume of the incubation system is 200 μl, which contains 1.3 mmol / l NADP+, 3.3 mmol / l G-6-P, 0.4 U / ml G-6-PDH, 3.3 mmol / l Mg 2+ , 2.5mmol / L UDPGA.

[0038] In this embodiment, the final volume of 200 μl has the following advantages: reaction efficiency: in a smaller volume, the concentration of reactants is relatively high, which facilitates rapid reaction. It is also convenient to operate; the smaller volume makes experimental operation easier and easier to control reaction conditions. It also saves materials, reduces the amount of reactants used, and reduces experimental costs.

[0039] Specifically, in step (d), the chromatographic column used was Waters UPLC HSS T3 (150×2.1 mm, 1.8 μm), the mobile phase A was 0.1% formic acid in water, B was 0.1% formic acid in acetonitrile, the gradient elution program was 0-1 min: 1% B, 1-8 min: 1% A-99% B, 8-10 min: maintain 99% B, 10-12 min: 1% B, and the flow rate was 0.3 mL / min.

[0040] In this embodiment, the Waters UPLC HSS T3 (150×2.1mm, 1.8μm) chromatographic column has high resolution and high sensitivity, capable of accurately separating and detecting individual components in complex mixtures. The 1.8μm particle size results in higher column efficiency and better separation, which facilitates more accurate identification and analysis of 4-HO-DET and its metabolites. The mobile phases of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) help improve the solubility and separation of the compounds. The addition of formic acid helps adjust the pH of the mobile phase and optimize the ionization state of the compounds, thereby improving separation and detection sensitivity.

[0041] Specifically, in step (d), the mass spectrometry conditions adopted a heatable electrospray ionization source (H-ESI), positive ion mode acquisition, nitrogen (N2) collision gas, injection voltage of 3800 V, vaporization temperature of 320 ° C, nebulizer gas pressure of 38 arb, auxiliary gas pressure of 15 arb, capillary temperature of 350 ° C, full scan acquisition data resolution of 35000, and secondary mass spectrometry acquisition data resolution of 17500.

[0042] In this embodiment, a heatable electrospray ionization source (H-ESI) is used. The H-ESI source heats the spray droplets, increasing the evaporation rate of the solvent, thereby enhancing the formation and transmission efficiency of ions. This helps increase the intensity and stability of the mass spectrometer signal and improve the sensitivity of detection. Positive ion mode acquisition is suitable for the detection of most basic or neutral compounds. If 4-HO-DET and its metabolites have appropriate ionization properties, they can be more effectively ionized and detected in positive ion mode.

[0043] Specifically, the phase I metabolites include hydroxylated metabolites, dihydroxylated metabolites, carboxylated metabolites, ketone-forming metabolites and dealkylated metabolites.

[0044] In this embodiment, understanding the metabolic pathways of 4-HO-DET in the human body through analysis of Phase I metabolites is crucial for understanding its biotransformation. Identifying metabolic markers, such as hydroxylation, dihydroxylation, carboxylation, ketone formation, and dealkylation products in Phase I metabolites, can serve as markers of 4-HO-DET metabolism and facilitate detection of its presence in biological samples.

[0045] Specifically, the phase II metabolite is a product of a hydroxylation reaction with glucuronic acid.

[0046] In this embodiment, Phase II metabolites, such as those resulting from hydroxylation followed by conjugation with glucuronic acid, generally have increased water solubility and polarity, which facilitates their excretion through urine or bile, thereby reducing the potential for accumulation in the body. This conjugation reaction can also reduce the biological activity of the metabolite, minimizing its potential interference with normal physiological functions or toxic effects in the body.

[0047] Specifically, the method also includes processing and analyzing mass spectrometry data using Mass Frontier software and Compound Discover software to deduce the structures of metabolites and identify unknown metabolites.

[0048] In this implementation, the accuracy of metabolite structure derivation is improved. MassFrontier software lists the possible fragments and fragmentation pathways of the parent drug, thereby facilitating the study of mass spectrometric fragmentation patterns. By simulating chemical reaction processes, it predicts the possible structural fragments of metabolites, providing important clues for subsequent structural derivation.

[0049] Specifically, this method can be used for metabolic detection, metabolic pathway analysis, and determination of metabolic markers in drug control work.

[0050] In this embodiment, the method improves detection accuracy by establishing an in vitro human liver microsome model to simulate the metabolic processes in the human body, enabling more accurate analysis of the metabolites of the tryptamine-type new psychoactive substance 4-HO-DET. This helps improve detection accuracy and helps drug control workers more accurately identify individuals who abuse these substances.

[0051] When using, 1. Experimental preparation

[0052] 1. Prepare the solution required for the reaction system:

[0053] NADPH regeneration system, potassium phosphate buffer, 4-HO-DET solution (prepared with DMSO and determine the appropriate concentration range for preliminary experiments), and liver microsome suspension (pay attention to the source, batch, and storage conditions of the liver microsomes)

[0054] 2. Prepare experimental equipment:

[0055] Centrifuge tubes, pipettes and tips, constant temperature oscillator or water bath, refrigerator, high performance liquid chromatography and corresponding detection columns

[0056] 2. Reaction System Preparation

[0057] 1. Operate on ice to avoid loss of enzyme activity.

[0058] 2. Add a certain volume of NADPH regeneration system and potassium phosphate buffer to each centrifuge tube.

[0059] 3. Add 4-HO-DET solution of specified concentration.

[0060] 4. Add liver microsome suspension to make the total volume of the reaction system reach the predetermined value.

[0061] 5. Use a pipette to gently mix to avoid creating bubbles.

[0062] 3. Reaction Process

[0063] 1. Place the prepared reaction system in a 37°C constant temperature oscillator or water bath and start timing.

[0064] 2. At predetermined time points (e.g., 0 min, 15 min, 30 min, 60 min, etc.), remove part of the reaction solution and place it on ice to terminate the reaction.

[0065] 3. The reaction solution should be immediately processed into the next step or stored in the refrigerator for testing.

[0066] 4. Sample processing and testing

[0067] 1. Add an appropriate amount of acetonitrile or other organic solvent to the sample after the reaction is terminated to precipitate the protein and extract the metabolites.

[0068] 2. Centrifuge the sample and take the supernatant for HPLC analysis.

[0069] 3. Calculate the amount of metabolites produced or the conversion rate based on the HPLC test results.

[0070] 5. Data Recording and Analysis

[0071] 1. Record the amount of metabolites produced or the conversion rate at each time point.

[0072] 2. Perform statistical analysis on the data and compare the effects of different concentrations of 4-HO-DET or different batches of liver microsomes on the metabolic rate.

[0073] 3. Based on the experimental results, draw relevant conclusions and provide reference for subsequent research.

[0074] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. Metabolites of the tryptamine-type new psychoactive substance 4-HO-DET identified in a human liver microsome model and their applications, including establishing an in vitro human liver microsome incubation model, characterized by: 4-HO-DET was incubated with mixed human liver microsomes at 37°C. An NADPH regeneration system was added to the incubation system to initiate the in vitro metabolic reaction. The incubation time was selected to be 90 minutes to ensure the acquisition of abundant metabolites. The metabolites were analyzed by ultra-performance liquid chromatography Q-Exactive tandem quadrupole-orbitrap high-resolution mass spectrometry. Data were acquired in Full MS-ddMS2 mode, and the structures of the metabolites were deduced based on the mass-to-charge ratios of the parent drug and fragment ions. At least seven metabolites were identified, including six phase I metabolites and one phase II metabolite. The main metabolic pathways were hydroxylation and conjugation with glucuronic acid after hydroxylation.

2. The metabolite of the tryptamine-type new psychoactive substance identified by the human liver microsome model 4-HO-DET and its use according to claim 1, characterized in that: The concentration of the mixed human liver microsomes was 20 mg / ml.

3. The metabolite of the tryptamine-type new psychoactive substance identified by the human liver microsome model 4-HO-DET and its use according to claim 1, characterized in that: The NADPH regeneration system includes NADP+, G-6-P, G-6-PDH, MgCl2 and UDPGA.

4. The metabolite of the tryptamine-type new psychoactive substance identified by the human liver microsome model 4-HO-DET and its use according to claim 1, characterized in that: The final volume of the incubation system was 200 μl, including 1.3 mmol / l NADP+, 3.3 mmol / l G-6-P, 0.4 U / ml G-6-PDH, 3.3 mmol / l Mg 2+ , 2.5mmol / L UDPGA.

5. The metabolite of the tryptamine-type new psychoactive substance identified by the human liver microsome model 4-HO-DET and its use according to claim 1, characterized in that: In step (d), a Waters UPLC HSS T3 (150×2.1 mm, 1.8 μm) column was used, mobile phases A and B were 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. The gradient elution program was as follows: 0-1 min: 1% B, 1-8 min: 1% A-99% B, 8-10 min: hold 99% B, 10-12 min: 1% B, and the flow rate was 0.3 mL / min.

6. The metabolite of the tryptamine-type new psychoactive substance identified by the human liver microsome model 4-HO-DET and its use according to claim 1, characterized in that: In step (d), the mass spectrometry conditions adopted a heatable electrospray ionization source (H-ESI), positive ion mode acquisition, nitrogen (N2) collision gas, spray voltage of 3800 V, vaporization temperature of 320°C, nebulizer gas pressure of 38 arb, auxiliary gas pressure of 15 arb, capillary temperature of 350°C, full scan acquisition data resolution of 35000, and secondary mass spectrometry acquisition data resolution of 17500.

7. The metabolite of the tryptamine-type new psychoactive substance identified by the human liver microsome model 4-HO-DET and its use according to claim 1, characterized in that: The phase I metabolites include hydroxylated metabolites, dihydroxylated metabolites, carboxylated metabolites, ketone-forming metabolites, and dealkylated metabolites.

8. The metabolite of the tryptamine-type new psychoactive substance identified by the human liver microsome model 4-HO-DET and its use according to claim 1, characterized in that: The phase II metabolite is a product of the reaction of hydroxylation with glucuronic acid.

9. The metabolite of the tryptamine-type new psychoactive substance identified by the human liver microsome model 4-HO-DET and its use according to claim 1, characterized in that: The method also includes processing and analyzing mass spectrometry data using Mass Frontier software and Compound Discover software to deduce the structures of metabolites and identify unknown metabolites.

10. The metabolite of the tryptamine-type new psychoactive substance identified by the human liver microsome model 4-HO-DET and its use according to claim 1, characterized in that: This method can be used for metabolic detection, metabolic pathway analysis, and determination of metabolic markers in drug control work.

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