WO2020159321A2 - Method for assessing medicinal effect of therapeutic agent for neuropsychiatric disorders by using microdialysis and simultaneous analysis of various neurotransmitters in primates - Google Patents

Abstract

The present application relates to a method for assessing a medicinal effect of a therapeutic agent for neuropsychiatric disorders in primates. The method for assessing a medicinal effect of a therapeutic agent for neuropsychiatric disorders disclosed in the present application comprises a microdialysis assay and analysis of various neurotransmitters.

Description

Neuropsychiatric drug treatment efficacy evaluation method using simultaneous analysis of microdialysis and multiple neurotransmitters in primates

The present invention relates to a method for evaluating the efficacy of a therapeutic agent for neuropsychiatric diseases in primates. More specifically, the present invention relates to a method of evaluating the efficacy of a therapeutic agent for a neuropsychiatric disorder targeting the central nervous system by collecting samples by microdialysis and analyzing multiple neurotransmitters simultaneously in a primate most similar to humans.

Almost all neuropsychiatric disorders, such as degenerative neuropathy and psychosis, are accompanied by disorders in neuromodulation. Its underlying pathological mechanism is caused by abnormalities in the control of the neurotransmitters.

Drugs developed to treat these neuropsychiatric disorders have been conducted in a number of preclinical and clinical trials. In most preclinical stages, rodents are used to confirm the effects of drugs, and in clinical stages, the effects of drugs are targeted to humans. Results in

Therefore, there is a need for a technique for evaluating the effectiveness of drugs for treating neurological diseases using primates with the most similar human and nervous systems.

One task of the present application is to provide a method for evaluating the efficacy of a therapeutic agent for neuropsychiatric diseases using simultaneous analysis of microdialysis and multiple neurotransmitters for primates.

Another task of the present application is to provide an experimental technique that can find biomarkers based on the variation of neurotransmitters for each type of neuropsychiatric disease and utilize them in drug efficacy evaluation of therapeutic agents for neuropsychiatric diseases.

Another task of the present application is to provide a non-clinical evaluation platform for the treatment of neuropsychiatric disorders by obtaining a microdialysis sample from a primate brain and simultaneously analyzing a variety of neurotransmitters using a mass spectrometer.

According to an embodiment of the present invention, in the method for evaluating the efficacy of a therapeutic agent for a neuropsychiatric disorder, a step of performing microdialysis against a primate, using a sample obtained as a result of the microdialysis, a plurality of neurotransmitters A method for evaluating drug efficacy for a treatment for a neuropsychiatric disease may be provided, comprising the step of simultaneously analyzing and evaluating a drug efficacy for the treatment for a neuropsychiatric disease based on the results of the simultaneous analysis.

According to another embodiment of the present invention, a method of using a primate as a target of microdialysis and collecting samples from several tissues at the same time may be provided.

According to an embodiment of the present application, by performing microdialysis and multiple neurotransmitters simultaneously on primates, an effect capable of evaluating the efficacy of a therapeutic agent for neuropsychiatric diseases may occur.

According to an embodiment of the present application, by finding a biomarker based on the variation of a neurotransmitter for each type of neuropsychiatric disease, an effect of providing an experimental technique that can be applied to drug efficacy evaluation of a therapeutic agent for neuropsychiatric diseases may occur.

According to an embodiment of the present application, by obtaining a microdialysis sample in the brain of a primate and simultaneously analyzing multiple neurotransmitters using a mass spectrometer, an effect of providing a nonclinical evaluation platform for a treatment for neuropsychiatric diseases may occur. .

1 to 4 is a flow chart for a method for evaluating the efficacy of a treatment for neuropsychiatric diseases using simultaneous micro-dialysis and multiple neurotransmitter analysis for primates according to an embodiment of the present application.

5 to 8 illustrate the results of analyzing neurotransmitters for primate brain-derived samples according to embodiments of the present application.

9 illustrates skull fixation using a primate brain stereotaxic target for a primate according to an embodiment of the present application.

FIG. 10 illustrates an example of a site for inserting a microdialysis probe for each primate brain tissue site for a primate according to an embodiment of the present application.

The above-described objects, features and advantages of the present application will become more apparent through the following detailed description in connection with the accompanying drawings. However, the present application may be modified in various ways and may have various embodiments. Hereinafter, specific embodiments will be illustrated in the drawings and described in detail.

Neural functions are regulated by 80 billion neurons in the brain and 100 trillion synapses, which are controlled by neurotransmitters and neuromodulators released into the synapse. It is practically impossible to identify this process in the human brain, and since rodents other than primates are not similar to the human and the nervous system is experimentally different, it is possible to confirm the free regulation of neurotransmitters and metabolites in the primate brain most similar to humans. Experimentation is essential.

Primates are the most similar animal groups in humans in many fields such as genetic, anatomical physiology, endocrine, skeletal, and behavioral patterns among all existing animals. There are 260 species of primates, which are classified into apes, old world monkeys, new world monkeys, and primitive monkeys. Among the world monkeys, red haired monkeys and crab monkeys are the most used species for experiments.

In Europe, there are primate research facilities managed by eight countries across the EU, including the United Kingdom, France, Independence, the Netherlands and Italy. In particular, the United States has eight primate centers, with an average of more than 2,000 primates per center.

In Japan, the Tsukuba Primate Center and the Kyoto Primate Center are leading primate research, and China is the largest exporter of research primate resources in the world, with the largest number of primate production facilities in the world and focusing on primate research. Doing.

In Korea, it is currently planning to expand to 3,000 heads, currently accepting about 500 heads at Jeongeup's National Primate Center. In addition, primates are being raised at the Safety Evaluation Research Institute, the Experimental Animal Center of Daegu and Osong Advanced Medical Complex, the Seoul National University Hospital, and the Design Animal Center of the Pyeongchang Campus of the Seoul National University.

It is very important to evaluate the effect of drugs on the free fluctuation of neurotransmitters in primates with similar human and nervous systems. In particular, it is ideal to accurately identify the causal circuit that is responsible for improving the understanding of the pathogenesis of mental or neurological diseases and to restore specific behavioral disorders or nerve damage caused by circuit abnormalities by regulating the neural circuit. . However, even for neuropsychiatric disorders and even normal neural circuit function, the circuit level is difficult to confirm by general experimental methods.

Recently, it is possible to selectively regulate neuronal activity by photogenetic methods, but since neuronal functions are regulated not only by the activity of specific genes, but also by interaction with glial cells around the nerve, the ultimate phenotype is related to neurotransmitters, neuropeptides and cytokines. It appears as a comprehensive action. They must be able to interpret them in an integrated manner, and the energy metabolism of the brain and the release of neurotransmitters show great differences depending on the level of stress even after death, anesthesia, waking state, and waking up. A suitable environment is to take samples while having a low-stress and daily life. The most appropriate method for these conditions is to use microdialysis for sampling.

The microdialysis is a microcirculation system by continuously injecting a circulating solution using a microdialysis pump in a state in which a probe with a dialysis membrane in the tissue to be measured is inserted. When it is maintained, the substance to be measured in or out of the body reaches an equilibrium state in which exchange is made between the circulating fluid and the body fluid. At this time, the body fluid is collected to analyze the concentration of the material.

That is, the microdialysis method is an experimental technique that can collect and measure in vivo and ex vivo substances through micropore of the dialysis membrane in extracellular fluid (ECF), which accounts for 15-20% of the total tissue fluid, The membrane that determines microdialysis is a semipermeable membrane that limits the molecular weight of the membrane, enabling simultaneous collection and measurement of all neurotransmitters of low molecular weight and can be continuously measured in the same animal.

When analyzing the microdialysis solution collected by the method using the microdialysis, an HPLC method is generally used, but a neurotransmitter difficult to analyze exists. However, recently, it was possible to analyze with much higher sensitivity using a mass spectrometer (LC-MS/MS).

This application is characterized by using simultaneous microdialysis and multiple neurotransmitter analysis for primates in order to increase the success rate of the development of therapeutic agents for neuropsychiatric disorders, which have a higher failure rate in clinical than other diseases.

In particular, the method of the present application is capable of simultaneously analyzing monoamines and amino acid-based neurotransmitters, as well as their metabolites, so it is also possible to evaluate the metabolic rate of neurotransmitters acting in neuropsychiatric disorders.

1 is a flow chart showing a method for evaluating the efficacy of a therapeutic agent for neuropsychiatric diseases using simultaneous analysis of microdialysis and multiple neurotransmitters for primates according to an embodiment of the present invention.

Referring to FIG. 1, a method for evaluating the efficacy of a treatment for a neuropsychiatric disorder using a simultaneous analysis of microdialysis and multiple neurotransmitters for primates is a step of performing microdialysis against primates (S1200). It may include the step of performing a simultaneous analysis of a plurality of neurotransmitters present in the obtained sample (S1400) and evaluating the drug efficacy based on the results of the simultaneous analysis (S1600).

Hereinafter, with reference to each drawing, each step of the method for evaluating the efficacy of a treatment for neuropsychiatric diseases using simultaneous micro-dialysis and multiple neurotransmitter analysis for primates will be described in more detail.

2 is a flowchart illustrating a method of performing microdialysis on a primate according to an embodiment of the present invention.

Referring to FIG. 2, the step of performing microdialysis against a primate (S1200) includes selecting a primate to be tested (S1220), anesthetizing the selected primate (S1240), and inserting a microdialysis probe into the selected primate It may include the step (S1260) and the step of obtaining a microdialysis sample using the inserted microdialysis probe (S1280).

Primates to be tested for obtaining a sample using microdialysis may be selected (S1220). Here, the selected primate may be, for example, an ape, an old world monkey, a new world monkey, or a primitive monkey, and more specifically, a rhesus monkey or a crab monkey may be selected.

Primates selected to insert a microdialysis probe may be anesthetized (S1240). Here, the anesthesia step may include, for example, general anesthesia, sleep anesthesia, or partial anesthesia.

A microdialysis probe may be inserted into a primate selected to obtain a sample through microdialysis (S1260). Here, a portion to which the microdialysis probe can be inserted may be determined. For example, a microdialysis probe can be inserted into the central nervous system, including the primate brain and spinal cord. When a microdialysis probe is inserted into the primate brain, the inserted portion may be each part of the brain including Superior frontal gyrus, Precentral gyrus, Postcentral gyrus, Posterior cingulate gyrus, Superior parietal lobule, Occipital gyrus.

For use in the analysis, a microdialysis sample can be obtained using the inserted microdialysis probe (S1280). Here, the sample can be obtained after a certain time after the microdialysis probe is inserted.

Figure 3 is a flow chart showing a method for performing a simultaneous analysis of a plurality of neurotransmitters present in a sample obtained by microdialysis.

Referring to Figure 3, performing a simultaneous analysis of a plurality of neurotransmitters present in a sample obtained by microdialysis (S1400) is a step of injecting the obtained microdialysis sample into the analysis device (S1420), the analysis device It may include the step of analyzing a variety of neurotransmitters contained in the sample using (S1440) and obtaining the analyzed results from the analysis device (S1460).

The obtained microdialysis sample can be injected into the analysis device (S1420). Here, the analytical device that can be used may be a device that can measure the concentration of substances contained in a sample. For example, the analytical instrument can be a mass spectrometer including LC-MS/MS or ELISA.

The analysis device may analyze a variety of neurotransmitters contained in the sample (S1440). Here, all substances used to transmit nerves may be included in the neurotransmitter. For example, neurotransmitters are GABA, Glutamate, Dopamine, Acethylcholine, Choline, Norepinephrine, Serotonin, 5-Hydroxyindoleacetic acid (5-HIAA), 3-Methoxy-4-hydroxyphenylglycol sulfate (MHPG-sulfate) and 3,4- Dihtdroxyphenylacetic acid (DOPAC). In addition, neurotransmitters include arginine, aspartate, glycine, D-serine, dopamine, epinephrine, histamine, and tyramine. , Octopamine, synephrine, tryptamine, N-methyltryptamine, anandamide, 2-Arachidonoylglycerol, 2- 2-Arachidonyl glyceryl ether, N-Arachidonoyl dopamine, Virodhamine, Adenosine, Adenosine triphosphate, Nicotinamide adenine It may include Nicotinamide adenine dinucleotide, encephalin, dynorphin, endorphin, endomorphin or nociceptin/orphanin FQ.

The analyzed result can be obtained from the analysis device (S1460). Here, the analyzed results may include the presence or absence of multiple neurotransmitters or concentration values of multiple neurotransmitters.

4 is a flow chart showing a method for evaluating drug efficacy based on the results of simultaneous analysis of multiple neurotransmitters.

Referring to Figure 4, the step of evaluating the drug efficacy based on the result of simultaneous analysis of the neurotransmitter (S1600), the step of confirming the analysis result of the microdialysis sample obtained before injecting the drug (S1620), the drug is injected It may include the step of confirming the analysis results of the microdialysis sample obtained after (S1640) and comparing the analysis results before and after drug injection (S1660).

Analysis results of the microdialysis sample obtained before the injection of the drug may be obtained (S1620). The drug used here may be a drug related to a change in a substance to be observed at a site where a microdialysis probe is inserted. For example, when the insertion site of the microdialysis probe is the brain in the central nervous system, the drug used may be a drug capable of changing the concentration of the neurotransmitter. For a specific example, the drugs used may include serotonin-norepinephrine reuptake inhibitors, and may include donepazil, which is used as a treatment for venlafaxine or Alzheimer's disease as an antidepressant.

Analysis results of the microdialysis sample obtained after the injection of the drug can be obtained (S1640). Here, the time to obtain a microdialysis sample may be a time point at which the drug starts to exhibit an effect after the drug is injected. In addition, a microdialysis sample obtained after injecting the drug may be obtained and obtained at least once.

The drug efficacy can be evaluated by comparing the analysis results before and after drug injection (S1660). Here, comparing the results of the analysis before and after drug injection is based on the difference between the concentration value of multiple neurotransmitters in the sample obtained before drug injection and the concentration value of multiple neurotransmitter concentrations in the sample obtained after drug injection. It is possible, but not limited, to compare the presence or absence of some neurotransmitters among a variety of neurotransmitters, and to compare based on ratios of concentration values of the obtained samples.

By comparing the analysis results before and after drug injection, changes in at least one neurotransmitter changed by the injected drug can be confirmed, and accordingly, efficacy of the drug on the at least one neurotransmitter can be evaluated.

5 to 8 is a graph showing the results of analyzing the microdialysis samples obtained according to the above-described drug evaluation method. That is, FIGS. 5 to 8 show a plurality of neurotransmitter analysis results for a microdialysis sample obtained after administration of venlafaxine to a crab monkey, and more details will be described below.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1. Experimental animals

A total of 10 cynomolgus monkeys (5 males, 5 females, 3-4 kg) were quarantined for about 1 month after ingestion, and passed through a purification period of at least 1 month before the start of the test in the animal room. The experimental group selected healthy individuals with no symptoms affecting the results. During the test period, individual breeding was carried out in a stainless steel breeding box (510WХ800LХ 764H, unit mm). The breeding environment was kept under the conditions of temperature 20-29°C, humidity 8-70%, illumination 12 hours day/night cycle, and illuminance 80-700 Lux. Feed was limited to approximately 120 g per day divided into morning and afternoon, and negative water was given freely. All animals were managed in accordance with the deliberation of the Institutional Animal Care and Use Committee (IACUC).

Example 2. Microdialysis of monkey brain

9 is a view showing that a primate skull is fixed using a primate brain stereotaxic device according to an embodiment of the present application.

Referring to FIG. 9, a brain stereotaxic fixture may be used to fix a primate skull when performing microdialysis.

10 is a diagram illustrating an example of a site for inserting a microdialysis probe for each primate brain tissue region for a primate according to an embodiment of the present application.

Referring to FIG. 10, a region on the primate brain for performing microdialysis may be provided.

Brain microdialysis in the present invention was carried out according to the following steps.

1) After sitting the monkey in a fixed cage for monkeys, induce respiratory general anesthesia with 4% isoflurane.

2) Move the anesthesia-induced monkey to the surgical table, maintain breathing with 2% isoflurane, and check that blood pressure, heart rate, and breathing are maintained in the normal range.

3) As a surgical dissector, the skull is incised in an oval shape from the frontal cortex to the occipital cortex.

4) Monkeys generalized with isoflurane fix their heads on a stereotaxic instrument (eg; see FIG. 9).

5) After removing the Dura membrane and arachinoid space, stop all bleeding sites.

6) For the microdialysis probe, a CMA 7 microdialysis probe from CMA was used, and the monkey brain cortex was divided into 7 regions (for example; see FIG. 10) to insert the probe. Microdialysis probes are inserted in the order of left and right superior frontal gyrus, precentral gyrus, postcentral gyrus, superior parietal lobule, and left and right occipital gyrus. Did.

7) To prevent the influx of body fluids and blood, a cloth consisting of a small grid was attached to the brain, and after confirming the insertion site, the microdialysis probe was inserted at an angle of about 1, 2 mm to a depth of about 45 degrees and surrounding it with a living body bond Was applied thinly. When the bond dries, apply it with dental cement to fix the bottom of the microdialysis probe. When the cement hardens, the microdialysis probe is inserted in the same way in the next area.

8) When the microdialysis probe is inserted into the 7 cortical regions, the inlet of the probe is connected to a syringe containing artificial cerebrospinal fluid, and then to a syringe pump. The outlet is placed in a 0.6 ml tube to receive the sample, and the flow rate of the syringe pump is set to 1.5 ul/min, and sampling is performed 14 times, 20 minutes per object. Immediately after inserting the probe, there may be a rapid change in concentration of neurotransmitters due to irritation. Collected samples are stored in a cryogenic freezer for analysis.

In this example, the microdialysis experiment was performed twice every 20 minutes in the basal state, 4 times in 1 hour increments after venlafaxine administration (5 males) or 2 times in 1 hour increments after venlafaxine administration, and in 1 hour increments after administration of donepezil. Two experiments (five females) were conducted.

Example 3. Sample administration

Venlafaxine hydrochloride was prepared by dissolving it in an appropriate concentration in physiological saline (0.9% NaCl). Venlafaxine hydrochloride is a serotonin-norepinephrine reuptake inhibitor that is used as an antidepressant. In addition, donepezil (Donepezil hydrochloride) was prepared by dissolving in physiological saline. Donepezil (Donepezil hydrochloride) is a drug used to treat Alzheimer's disease. The prepared samples were injected intravenously at a dose of 2mg/kg/1ml in venlafaxine to crab monkeys 40 minutes after the start of microdialysis sampling, and donepezil was injected intravenously in a dose of 500µg/kg/1ml. .

Example 4. Analysis method (LC-MS/MS)

As the LC used in the present invention, ACQUITY UPLC I-Class PLUS System of Waters and MS/MS were composed of Triple Quadrupole 6500+ System of AB SCIEX, and the ionization method of MS was electrospray ionization (ESI). Positive and negative ions were analyzed simultaneously using the method. Ion separation was analyzed by reverse-phase liquid chromatography using LC-MS/MS with a triple quadrupole mass separation tube attached.

As a separation condition, an ACQUITY UPLC HSS T3 (2.1Х100 mm, 1.8 μm, Waters) column was used, and the mobile phase contained an aqueous solution (A) containing 0.1% Formic acid and 5 mM Ammonium formate in HPLC Water and Methanol and Acetonitrile 1: Using the aqueous solution (B) containing 5 mM Ammonium formate in the solution mixed with 1, B was started at a composition of 0.5% based on the initial starting 0 minutes and maintained until 0.5 minutes, and composition of B was 5% in 1 minute. By changing the gradient composition to 40% until 2 minutes and B to 90% at 2.5 minutes, the gradient composition was maintained at 90% until 4.5 minutes. In 4.6 minutes, B was flowed up to 7.5 minutes with 0.5% composition to stabilize at the initial composition ratio. The flow rate was set at 0.3 mL/min. The column temperature was maintained at 25°C, and samples were injected at 10 µl each. The conditions were used in the same manner as in Table 1 (LC analysis conditions).

Instrument	Waters ACQUITY UPLC I-Class PLUS System
Column	Waters ACQUITY UPLC HSS T3 (2.1 x 100 mm, 1.8 μm)
Mobile phase (A)	Water (0.1% Formic acid, 5 mM Ammonium formate)
Mobile phase (B)	MeOH: Acetonitrile = 1:1 (5 mM Ammonium formate)
Gradient	Time (min)	A%	B%
	0.0	99.5	0.5
	0.5	99.5	0.5
	1.0	95.0	5.0
	2.0	60.0	40.0
	2.5	10.0	90.0
	4.5	10.0	90.0
	4.6	99.5	0.5
	7.5	99.5	0.5
Column Temp	25℃
Injection volume	10 μl
Flow rate	0.3 mL/min

Mass spectrometer was analyzed by ESI and nitrogen was used for drying gas. It was operated under MRM (Multiple Reaction Monitoring), a multi-component simultaneous analysis method. Both positive and negative ion spray voltages were fixed at 4500 V. Curtain gas (CUR), Collision gas (CAD), Ion source gas 1 (GS1), and Ion source gas 2 (GS2) were maintained at 30, medium, 50, and 60, respectively, and were fixed at ion transfer tube temperaturesms of 550°C. The conditions were used in the same manner as in Table 2 (MS/MS analysis conditions).

Instrument	AB SCIEX Triple Quadrupole 6500+
Ion Source Type	ESI
Positive Ion Spray Voltage (V)	4500
Negative lon Spray Voltage (V)	-4500
Curtain gas (CUR)	30
Collision gas (CAD)	Medium
Ion source gas 1 (GS1)	50
Ion source gas 2 (GS2)	60
Ion Transfer Tube Temp (℃)	550

The chromatogram for the analysis result of the brain neurotransmitter is as shown in FIG. 2. In the above, the configuration and features of the present application have been described based on the embodiment according to the present application, but the present application is not limited thereto, and the spirit of the present application It is apparent to those skilled in the art to which the present application pertains to various changes or modifications within the scope and scope, and thus, it is revealed that such changes or modifications belong to the appended claims.

The form for carrying out the present invention may include the best form for carrying out the above-described invention, and related matters are described in the best form for carrying out the invention.

Claims (13)

1. A method of using primates as targets for microdialysis and collecting samples from multiple tissues simultaneously.
2. After primate anesthesia, skull fixation using a primate stereotaxic instrument to locate the microdialysis probe on a specific target site and fix it so that it does not move during the test.
3. A method of fixing a microdialysis probe to brain tissue by attaching a cloth made of a small grid to the brain to prevent the influx of body fluids and blood, inserting a microdialysis probe, and thinly applying the surroundings with a bio-bond.
4. The method of claim 2, further comprising the step of stably fixing the lower portion of the microdialysis probe to the cerebral cortex with a dental cement.
5. A method of causing free fluctuations of neurotransmitters and metabolites by drug administration after the microdialysis probe is fixed and stabilized.
6. Simultaneous analysis of neurotransmitters (GABA, glutamate, acetylcholine, norepinephrine, dopamine, serotonin) and metabolites (choline, MHPG, MHPG sulfate, 5-HIAA, DOPAC) by mass spectrometry in samples obtained by microdialysis.
7. In the method of evaluating the drug efficacy for the treatment of neuropsychiatric diseases,

   Performing microdialysis against a primate;

   Simultaneously analyzing a plurality of neurotransmitters using a sample obtained as a result of the microdialysis; And

   Comprising the steps of evaluating the drug efficacy for the treatment of neuropsychiatric diseases based on the results of the simultaneous analysis;

   Neuropsychiatric drug treatment method.
8. The method of claim 7,

   The site where the microdialysis is performed is two or more selected from Superior frontal gyrus, Precentral gyrus, Postcentral gyrus, Posterior cingulate gyrus, Superior parietal lobule or Occipital gyrus,

   Neuropsychiatric drug treatment method.
9. The method of claim 7,

   The plurality of neurotransmitters to be analyzed simultaneously is GABA, Glutamate, Dopamine, Acethylcholine, Choline, Norepinephrine, Serotonin, 5-Hydroxyindoleacetic acid (5-HIAA), 3-Methoxy-4-hydroxyphenylglycol sulfate (MHPG-sulfate) or 3 ,4-Dihtdroxyphenylacetic acid (DOPAC) two or more substances selected from,

   Neuropsychiatric treatment drug efficacy evaluation method
10. The method of claim 7,

    The neuropsychiatric disorder is depression or Alzheimer's,

    Neuropsychiatric drug treatment method.
11. The method of claim 7,

    The step of performing a microdialysis on the primate,

    Selecting the primate;

    Anesthetizing the primate;

    Inserting a microdialysis probe into the primate; and

    Obtaining a microdialysis sample; comprising

    Neuropsychiatric drug treatment method.
12. The method of claim 7,

    Simultaneously analyzing a plurality of neurotransmitters using a sample obtained as a result of the microdialysis,

    Injecting the obtained sample into an analytical instrument;

    Analyzing the plurality of neurotransmitters using the analysis device; and

    Acquiring the analyzed result from the analysis device; containing

    Neuropsychiatric drug treatment method.
13. The method of claim 7,

    Evaluating the drug efficacy for the treatment of neuropsychiatric diseases based on the results of the simultaneous analysis,

    Confirming an analysis result of the microdialysis sample obtained before injecting the drug;

    Confirming an analysis result of the microdialysis sample obtained after injecting the drug; And

    Comprising the step of evaluating the drug efficacy by comparing the analysis results before and after the drug injection

    Neuropsychiatric drug treatment method.

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