WO2024061250A1 - Diagnostic biomarker for depression and use thereof - Google Patents

Abstract

Disclosed is the use of a compound 4-TMAP as a diagnostic biomarker for depression. It has been found in the present invention that the difference in serum or feces 4-TMAP between patients with depression and a healthy population is statistically significant. By means of analyzing the relative 4-TMAP content in the serum or feces of a patient to be tested, it is possible to easily indicate whether the patient suffers from depression, or indicate a potential risk of depression. The detection method of the present invention is simple and convenient, is convenient for use in physical examinations and large-scale screening, is conducive to early detection and early treatment, and has broad market prospects.

Description

A biomarker for depression diagnosis and its application Technical field

The present invention relates to the field of disease diagnosis, and in particular to a biomarker related to the pathogenesis of depression and its application.

Background technique

Depression (MDD) is now the most common mental illness, with continuous and long-term low mood as the main clinical feature. It is the most important type of mental illness in modern people. Clinically, it can be seen that people are depressed and unhappy in reality, and their mood has been low and depressed for a long time. From being unhappy at the beginning to being distraught at the end, they have low self-esteem, pain, pessimism, and world-weariness. They feel that they are torturing themselves in despair every day of their lives. , negativity, avoidance, and finally even suicidal tendencies and behaviors. The patient suffers from somatization symptoms. Chest tightness and shortness of breath.

Depression (MDD) is a common disabling disease with high incidence in the world and has a significant impact on patients' physical and mental health. Depression can impair patients' ability to work and study, seriously affect their quality of life, and may lead to suicide. A 2017 meta-analysis on the Global Burden of Disease Study noted that depression has affected approximately 300 million people and is one of the leading causes of disability and disease burden around the world.

The diagnosis of depression should mainly be based on medical history, clinical symptoms, disease course, physical examination and laboratory tests. The diagnosis of typical cases is generally not difficult. The internationally accepted diagnostic standards generally include ICD-10 and DSM-IV. In China, ICD-10 is mainly used, which refers to the first episode of depression and recurrent depression, excluding bipolar depression. Patients often have typical symptoms such as depressed mood, loss of interest and pleasure, low energy, or feelings of fatigue. Other common symptoms are (1) reduced ability to focus and pay attention; (2) reduced self-esteem; (3) thoughts of guilt and worthlessness (even in mild attacks); (4) pessimistic view of the future ; (5) Ideas or behaviors of self-harm or suicide; (6) Sleep disorders; (7) Decreased appetite.

Correct diagnosis, especially early diagnosis of depression, is crucial for effective treatment; however, the diagnosis of depression is still based on clinical symptomatology criteria so far. In addition, the clinical symptoms of depression are diverse and lack specificity, so the misdiagnosis rate is high and hinders effective treatment. Unfortunately, although researchers have been working on this in recent decades and have discovered many biomarkers related to the pathogenesis of depression, there is still a lack of effective diagnostic markers that can be used clinically.

Contents of the invention

The main purpose of the present invention is to provide a biomarker related to depression, which can effectively solve the problems in the background technology.

To achieve the above object, the technical solution adopted by the present invention is:

Provided is the use of compound 4-TMAP represented by formula (I) as or preparing a biomarker for depression diagnosis. The structure of said compound 4-TMAP is: (I).

Further, the present invention provides a detection kit, which contains compound 4-TMAP and necessary diluent; preferably, the 4-TMAP and diluent can be configured to (3-10) ng/mL The concentration is preferably 4-8 ng/mL, more preferably 5 ng/mL; the preferred diluent includes water.

Further, the present invention provides an early warning system for depression. The early warning system includes the detection kit and analysis tools. Preferably, the analysis tools include high performance liquid chromatography (HPLC) or high performance liquid chromatography-mass spectrometry. (HPLC-MS).

Furthermore, the present invention provides a method for diagnosing depression, which comprises the step of determining the 4-TMAP content in the serum or feces of a human or animal to be tested; preferably, a prompt or warning is given when the 4-TMAP content is ≥5 ng/mL.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention discovers for the first time the statistical difference in the relative content of 4-TMAP in the feces and serum of healthy people and patients with depression, and determines the use of 4-TMAP as a biomarker for the diagnosis of depression.

The method of the present invention can be used for early screening of patients with depression or potential depression patients conveniently and quickly, and the risk of depression can be found, which is conducive to early detection and early treatment, and prevents the deterioration of the condition of potential depression patients.

Description of the drawings

Figure 1: Relative content of 4-TMAP in serum samples of patients with depression (MDD) and healthy people (HC);

Figure 2: Relative content of 4-TMAP in fecal samples of patients with depression (MDD) and healthy people (HC);

Figure 3: Relative content of 4-TMAP in serum samples of normal food group and amino acid food group in AFD-induced mouse depression model;

Figure 4: Relative content of 4-TMAP in fecal samples of normal food group and amino acid food group in AFD-induced mouse depression model;

Figure 5: Relative content of 4-TMAP in serum samples of normal mice and CRS depressed mice in the CRS-induced mouse depression model;

Figure 6: Relative content of 4-TMAP in fecal samples of normal mice and CRS depressed mice in CRS-induced mouse depression model;

Figure 7: Relative content of 4-TMAP in serum samples of normal mice and Cort-depressed mice in the Cort-induced mouse depression model;

Figure 8: Relative content of 4-TMAP in fecal samples of normal mice and Cort-depressed mice in the Cort-induced mouse depression model;

Figure 9: Distribution of 4-TMAP in intestinal and whole brain samples of normal mice, AFD, CRS, and Cort-induced mouse depression models.

Implementation

In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention will be further elaborated below in conjunction with specific implementation modes.

Example 1: 4-TMAP can be used as a clinical diagnostic marker for depression

Feces and serum samples were collected from patients with depression (MDD) and healthy people (HC), and the levels of 4-TMAP were detected by HPLC-MS mass spectrometry. The specific method is as follows:

(1) Sample processing and extraction:

Take 100 μL serum sample, add acetonitrile to adjust the volume to 1 mL, vortex for 1 min, mix and centrifuge at 12000 r/min for 10 min. Take the supernatant solution, pass it through a 0.22 μm filter membrane, and inject the sample into HPLC-MS.

Take 100 mg of fecal sample, add acetonitrile to 1 mL, vortex for 1 min, mix well, and centrifuge at 12000 r/min for 10 min. Take the supernatant solution, filter it through a 0.22 μm filter membrane, and inject it into HPLC-MS.

Preparation of standard material solution: Take the standard material mother solution, add water to prepare standard material solutions with concentrations of 1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, and 100ng/mL respectively, and cross 0.22 μm Filter membrane, HPLC-MS/MS analysis.

(2) The detection parameters are as follows:

HPLC parameters:

Liquid chromatography model: LC30

Column: Waters ACQUITY UPLC HILLC (100mm*2.1mm, 1.7µm)

Mobile phase: A: 0.1% formic acid aqueous solution Column temperature: 35°C

B: Methanol Flow rate: 0.30mL/min

Injection volume: 1 µL

Gradient elution procedure:

0.01	Pump	B.Conc	0
2.00	Pump	B.Conc	10
3.00	Pump	B.Conc	90
3.50	Pump	B.Conc	10
5.00	controller	stop

Mass spectrometry conditions:

Mode: ESI, positive ion mode analysis, scanning mode: multiple reaction monitoring (MRM), drying gas: nitrogen 10.0L/min, heating gas: air 10.0L/min, collision gas: argon 270kPa, atomizing gas: 3.0L /min, interface temperature: 200℃, DL tube temperature: 280℃, dwell time: 150ms, heating module temperature: 300℃, delay time: 3ms, interface voltage: 1.0KV,

Q1 and Q3 are both unit resolutions

Liquid quality instrument model: Shimadzu LC-MS-MS-8050

Table 2 Multiple reaction monitoring mode (MRM) parameters

Tab.2 Multiple reaction monitoring(MRM) conditions

Test conclusion:

The results are shown in Figures 1 and 2. The level of 4-TMAP in serum samples of MDD (n=142) was significantly lower than that of HC (n=49), with statistical significance; the level of 4-TMAP in fecal samples of MDD (n=114) The level of TMAP was significantly lower than that of HC (n=47), suggesting that 4-TMAP has the potential to be used as a clinical diagnostic marker for depression.

When the feces or serum samples of the patients to be tested are processed and tested by the above methods, and the content of 4-TMAP is 5 ng/mg or below 5 ng/ml, it indicates a higher risk of depression and the need for further evidence based on other methods. Retest to determine if you have depression.

Example 2 : 4-TMAP is significantly reduced in AFD-induced mouse depression model

Six 2-day-old SPF grade C57/B6j male mice (purchased from Jicui Yaokang) were randomly divided into 2 groups, one was the normal food group (NCD, 3 mice), and the other was the amino acid food group (AFD, 3 mice). 3), each group was assigned 1 nursing female mouse, and the female mice ate the corresponding food and then suckled the mice. After four weeks of continuous feeding, the female rats were separated, and the amino acid food group was replaced with normal food. Their depression status was monitored through forced swimming, tail suspension, and sugar water preference experiments.

The serum, feces, whole brain and intestinal tissues of NCD and AFD mice were taken respectively, and the levels of 4-TMAP were detected by HPLC-MS.

The processing and detection methods of serum and stool are the same as those of patient samples. The test results are shown in Figure 3 and Figure 4. 4-TMAP was significantly reduced in serum and feces samples of the AFD-induced mouse depression model.

Detection method of whole brain and intestines: Extract fresh whole brains and intestines from mice, quickly freeze them in liquid nitrogen, freeze sections, 10um, and use imaging mass spectrometry to detect the distribution of 4-TMAP.

Example 3 : 4-TMAP is significantly reduced in the CRS-induced mouse depression model

Six 6-8 week old SPF grade C57/B6j male mice (purchased from Jicui Yaokang) were randomly divided into 2 groups, one group was normal mice group (3 mice), and the other group was CRS mice (3 mice). Only). After adapting to the environment, starting from day 0, the CRS mouse group received chronic, unpredictable mild stimulation for 21 days, simulating the chronic low-intensity stress that humans receive in daily life. Every day, mice were treated with one stimulus from day and night, in random order and without repetition, so that the mice could not predict the appearance of the stimulus. After 21 days of continuous modeling, the success of depression modeling was confirmed through forced swimming, tail suspension, and sugar water preference experiments (significant differences from normal mice).

The serum, feces, whole brain and intestinal tissues of NCD and CRS mice were taken respectively, and the levels of 4-TMAP were measured by HPLC-MS.

The processing and detection methods of serum and feces are the same as those of patient samples. As shown in Figures 5 and 6, 4-TMAP is significantly reduced in serum and fecal samples of the CRS-induced mouse depression model.

Detection method of whole brain and intestines: Extract fresh whole brains and intestines from mice, quickly freeze them in liquid nitrogen, freeze sections, 10um, and use imaging mass spectrometry to detect the distribution of 4-TMAP.

Example 4 : 4-TMAP is significantly reduced in Cort-induced mouse depression model

Six 6-8 week old SPF grade C57/B6j male mice (purchased from Jicui Pharmaceutical) were randomly divided into two groups, one group was a normal mouse group (3 mice) and the other group was a Cort group (3 mice). After adapting to the environment, from day 0, the drinking water of the Cort mouse group (MCE, HY-B1618) was a corticosterone solution with a final concentration of 25μg/mL (pH 7.0-7.4), and the normal mouse group received ordinary drinking water, which was changed every 2 days, and the model was established for 21 days. After the model was established, the forced swimming, tail suspension, and sugar water preference tests were used to confirm the success of the model (there was a significant difference from normal mice)

The serum, feces, whole brain and intestinal tissues of NCD and Cort mice were taken respectively, and the levels of 4TMAP were detected by HPLC-MS.

The processing and detection methods of serum and feces are the same as those of patient samples. The detection results are shown in Figures 7 and 8, and 4-TMAP is significantly reduced in serum and feces samples of the Cort-induced mouse depression model.

Detection method of whole brain and intestines: Extract fresh whole brains and intestines from mice, quickly freeze them in liquid nitrogen, freeze sections, 10um, and use imaging mass spectrometry to detect the distribution of 4-TMAP.

The imaging mass spectrometry results are shown in Figure 9. 4-TMAP was significantly reduced in intestinal and whole brain samples of AFD, CRS, and Cort-induced mouse depression models.

The basic principles and main features of the present invention and the advantages of the present invention have been shown and described above. Those skilled in the industry should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will also have other aspects. Various changes and modifications are possible, which fall within the scope of the claimed invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims (10)

1. Use of compound 4-TMAP represented by formula (I) to prepare biomarkers for depression diagnosis. The structure of compound 4-TMAP is (I).
2. A detection kit, characterized in that the kit contains the biomarker according to claim 1.
3. The detection kit according to claim 2, characterized in that the detection kit also contains necessary diluents.
4. The detection kit according to claim 3, characterized in that the biomarker and diluent can be configured to a concentration of 3-10 ng/mL.
5. The detection kit according to claim 3, characterized in that the biomarker and diluent can be configured to a concentration of 4-8 ng/mL.
6. The detection kit according to claim 3, characterized in that the biomarker and diluent can be configured to a concentration of 5 ng/mL.
7. The detection kit according to any one of claims 3 to 6, characterized in that the diluent is selected from water.
8. A depression early warning system, characterized in that the early warning system comprises the detection kit and analysis tool according to any one of claims 2-7.
9. The depression early warning system according to claim 8, characterized in that the analysis tool is selected from high performance liquid chromatography (HPLC).
10. The depression early warning system according to claim 8, characterized in that the analysis tool is selected from high performance liquid chromatography-mass spectrometry (HPLC-MS).