CN1552331A - Compound 7-Carbonylepiandrosterone Improves Depression in Chronic Mild Stress BALB/C Mice - Google Patents

Abstract

The present invention relates to a synthetic compound 7-carbonyl dehydroepiandrosterone. The compound can increase the drinking amount of sucrose solution of depressed mice in the animal model of chronic mild stress depression, increase the spontaneous activity of depressed mice, and has antidepressant effect. In addition, 7-carbonyl dehydroepiandrosterone can enhance the immune function caused by depression and improve the learning and memory function of depressed mice. 7-Carbonyl dehydroepiandrosterone can be used as a candidate drug for depression and depression-induced immune dysfunction and learning and memory decline.

Description

Compound 7-Carbonylepiandrosterone Improves Depression in Chronic Mild Stress BALB/C Mice

Technical field:

The invention studies the improvement effect of 7-carbonyl dehydroepiandrosterone on depression by establishing a chronic mild stress depression model, and relates to the field of development and application of new drugs in biomedical high technology.

Background of the invention:

The World Health Organization once published a survey on the "burden of disease", based on the "disability" caused by disease - functional impairment statistics, depression ranked second, second only to chronic obstructive pulmonary disease, accounting for all diseases 6.2% of the total burden; if the 13th-ranked bipolar disorder (manic-depressive alternate episodes) is added, it will reach 8.0%. With the process of modernization, the importance of depression will continue to increase, and it is estimated that after 25 years, it will account for about 10% of the total disease burden. In a sense, depression will be a challenge across the century. In the pathogenesis of anxiety and depression, it is believed that the abnormal biochemical function of the central nervous system is closely related to it, and mainly involves neurotransmitters such as NE and 5-HT. As early as the 1950s, monoamine oxidase inhibitors (MAOIS) were used in the treatment of depression, but they were replaced by tricyclic antidepressants (TCAS) because of their severe side effects. TCAS has better curative effect and is still used today. . However, clinical findings show that TCAS still has many adverse reactions that make patients unbearable and discontinue use. At the same time, they have no effect on the low immune function and learning and memory dysfunction associated with depression. Therefore, it is the goal pursued by psychopharmacologists to choose compounds with less toxic and side effects, good therapeutic effect and can improve the comorbidities of depression.

Purpose of the invention:

By using a variety of stressors arranged in an unpredictable way on BALB/C mice, on the basis of establishing an animal model similar to human depression, the effect of 7-carbonyl dehydroepiandrosterone on depressive symptoms in depressed mice was studied Improvement effect, and evaluate the improvement effect of 7-carbonyl dehydroepiandrosterone on depression complicated immune function and learning and memory impairment, and lay the theoretical and technical foundation for the clinical application of 7-carbonyl dehydroepiandrosterone.

Content and requirements:

Establish an animal model of chronic mild stress depression, add 7-carbonyl dehydroepiandrosterone to the depressed mice, measure the changes in the drinking amount of 2% sucrose solution in the mice, and observe the effect of 7-carbonyl dehydroepiandrosterone on reversing the euphoria caused by depression. Pleasure situation. At the same time, the effect of 7-carbonyl dehydroepiandrosterone on the spontaneous activity of depressed mice was observed. Combined with the analysis of lymphocyte proliferation assay and natural cell killing (NK) assay, the effect of 7-carbonyl dehydroepiandrosterone on immunosuppression caused by depression was evaluated. The Morris water maze method was used to evaluate the effect of 7-carbonyl dehydroepiandrosterone supplementation on the learning and memory ability of depressed mice.

The technical index that this compound reaches is:

1. Supplementation of 7-carbonyl dehydroepiandrosterone can restore the reduced drinking volume of 2% sucrose solution in depressed mice, and improve the symptoms of euphoria in depressed mice.

2. Supplementation of 7-carbonyl dehydroepiandrosterone can reverse the decrease of spontaneous activity in depressed mice and restore the spontaneous activity of mice.

3. Supplementation with 7-carbonyl dehydroepiandrosterone reversed the hypoimmune function of mice caused by depression, and enhanced the immune function of depressed mice.

4. After supplementation with 7-carbonyl dehydroepiandrosterone, the spatial learning and memory ability of depressed mice can be improved, and the learning ability of depressed mice can be enhanced.

Description of drawings:

Figure 1. The structural formula of 7-carbonyl dehydroepiandrosterone

Molecular formula C ₁₉ H ₂₇ O ₃ , molecular weight: 302.4

Figure 2. The effect of 7-carbonyl dehydroepiandrosterone on the consumption of 2% sucrose solution in depressed mice

Control is the control group, Stress is the model group, Stress+7OD-L is the 5mg/kg dosage group, and Stress+7OD-H is the 15mg/kg dosage group.

Figure 3. Effect of 7-carbonyl dehydroepiandrosterone on locomotor activity in depressed mice

Control is the control group, Stress is the model group, Stress+7OD-L is the 5mg/kg dosage group, and Stress+7OD-H is the 15mg/kg dosage group.

Figure 4. The effect of 7-carbonyl dehydroepiandrosterone on spatial learning and memory in depressed mice

Control is the control group, Stress is the model group, Stress+7OD-L is the 5mg/kg dosage group, and Stress+7OD-H is the 15mg/kg dosage group.

Figure 5. The effect of 7-carbonyl dehydroepiandrosterone on the proliferation response of T lymphocytes stimulated by Con A in depressed mice

Control is the control group, Stress is the model group, Stress+7OD-L is the 5mg/kg dosage group, and Stress+7OD-H is the 15mg/kg dosage group.

Figure 6. The effect of 7-carbonyl dehydroepiandrosterone on the killing activity of NK cells in depressed mice

Control is the control group, Stress is the model group, Stress+7OD-L is the 5mg/kg dosage group, and Stress+7OD-H is the 15mg/kg dosage group.

Example:

1. Synthetic preparation of 7-carbonyl dehydroepiandrosterone

DHEA (10 g, 0.0255 mol) was dissolved in a mixed solution of 12 ml pyridine and 8 ml acetic anhydride, and stirred overnight at room temperature. The progress of the reaction was monitored by TLC (developer: ethyl acetate:petroleum ether=1:3). After 254nm ultraviolet detection, it was infiltrated with 5% sulfuric acid/ethanol solution and heated to develop color. (DHEA and 3β-Acetoxyandrost-5-ene-17-one have no fluorescence, but can develop color.) Pour the reaction mixture into 100ml ice water, adjust the pH to 6-7 with 2M HCl solution, and collect the precipitate by suction filtration. The solid was washed several times with ice water until the filtrate was neutral. Dry in an infrared lamp box for 10 hr. 10.5 g of white solid was obtained, m.p. 171-173°C, yield 92%. Recrystallization (acetone/petroleum ether) gave white crystals, m.p.172-173°C.

Install the reflux tube, condenser tube and ventilation tube in the there-necked flask, dissolve 2.5g of N-hydroxyphthalimide in a 55ml mixed solution of acetone/ethyl acetate (1:1), add 3β-acetate oxygen DHEA 5g and azocyclohexyl 0.5g. The reaction was heated to reflux, and the air was continuously introduced, and azocyclohexyl was added in batches to the reaction (about 50 mg×15 times each). After reacting for 50 hours, the solvent was evaporated to dryness. Add 40 ml of toluene, stir at 50°C for 1 hour, and then let it cool to room temperature. The solid (N-hydroxyphthalimide) was removed by filtration and reused and washed with a little toluene. The filtrate was washed with saturated aqueous sodium bicarbonate until the aqueous layer was colorless. Toluene was removed under reduced pressure. Purify by silica gel chromatography (column: 5×15 cm, developing solvent: ethyl acetate:petroleum ether=1:4) and collect the filtrate section containing the product to obtain 1.5 g of crude product. Purified by column chromatography (column: 20 × 3 cm, eluent petroleum ether: ethyl acetate = 4: 1), collected the eluate, concentrated to obtain 220 mg of light yellow solid. m.p. 185-187°C, yield 31%. Recrystallization from acetone/methanol gave white crystals with a melting point of 186-187°C.

About 2g of the steroid was dissolved in 90ml of methanol, to which 500mg of potassium hydroxide was added in an aqueous solution dissolved in 2.5ml, and the reaction solution was stirred at room temperature for 1 hour (TLC developer petroleum ether: ethyl acetate=3:2). The methanol was removed under reduced pressure to half the volume, poured into 100ml of water, placed at 0°C for about 1 hour, the solid was collected by filtration and washed with water several times to obtain 1.7 g of dry solid. The melting point was detected as 212-217°C.

Recrystallization Dissolve the solid in chloroform and filter once with cotton. The filtrate was collected and concentrated, crystals were precipitated after adding petroleum ether, the solid was collected and washed with a small amount of chloroform: petroleum ether about 1:5, and the solid was dried to obtain 1.55 g of needle crystals. m.p.219-222°C. The structure is shown in Figure 1.

2. Preparation of 7-carbonyl dehydroepiandrosterone suspension

Weigh 7-carbonyl dehydroepiandrosterone and prepare two suspensions of 2 mg/ml and 6 mg/ml with sesame oil (product of Sigma Company).

3. Establish an animal model of chronic mild stress depression

The present invention adopts the mouse stress program established by Monleon et al. and slightly changes it. The weekly stress program includes the following stressor content: food deprivation twice, each 12h; drinking water deprivation twice, each 14h; twice overnight Illumination; twice feeding in wet cages, 14 hours each time; swimming in ice water twice, 5 minutes each time; hanging upside down twice, 30 minutes each time; pair breeding twice, 17 hours each time. The above-mentioned stressors were randomly combined and implemented, and two kinds of stressors were treated every day for 4 consecutive weeks. Control mice were also housed in the same environment, but without exposure to treatment animals and stressors.

4. Supplementation of animals with 7-carbonyl dehydroepiandrosterone suspension

The stressed animals were divided into three groups: model group, low dose of 7-carbonyl dehydroepiandrosterone (5 mg/kg), high dose of 7-carbonyl dehydroepiandrosterone (15 mg/kg). 7-carbonyl dehydroepiandrosterone low dose, 7-carbonyl dehydroepiandrosterone high dose three groups of mice were injected intraperitoneally with 2 mg/ml or 6 mg/ml 7-carbonyl dehydroepiandrosterone at 8:00 every morning at 5 ml per kg body weight Androsterone suspension; the animals in the control group and the model group were intraperitoneally injected with sesame oil instead of the 7-carbonyl dehydroepiandrosterone suspension.

5. The effect of 7-carbonyl dehydroepiandrosterone on the drinking amount of 2% sucrose solution in depressed mice

After 4 weeks of stress treatment, the mice were fasted for 3 hours, and then the drinking water was replaced with 2% sucrose solution. The drinking amount of 2% sucrose solution was measured within 1 hour and corrected by body weight. 7-carbonyl dehydroepiandrosterone can increase the consumption of sucrose solution in depressed mice and reverse the loss of euphoria caused by depression (Figure 2).

6. Effect of 7-carbonyl dehydroepiandrosterone on locomotor activity in depressed mice

After 4 weeks of chronic stress treatment, the BALB/C mice of each group were placed in the mouse spontaneous activity box (developed by the Institute of Materia Medica, Chinese Academy of Medical Sciences) in a quiet environment, and the effect of 7-carbonyl dehydroepiandrosterone on the mice was observed. Effects on locomotor activity in depressed mice. 7-carbonyl dehydroepiandrosterone can increase spontaneous activity in depressed mice (Fig. 3).

7. Effects of 7-carbonyl dehydroepiandrosterone on spatial learning and memory in depressed mice

The water maze test was performed after the locomotor activity measurement. The water maze device consists of a cylinder with a diameter of 90 cm and a motion video capture system (product of Beijing Jierio Technology Co., Ltd.). Before the experiment starts, pour clear water into the bucket, the water depth is 15cm, adjust the water temperature to 20°C, then place a 7cm-diameter circular solid table in the bucket, the tabletop is about 1cm full of water, and use carbon ink to darken the water to prevent animals from seeing it. Real Taiwan. Start the water maze test program, calibrate the capture field of view, real platform, virtual platform and water entry point and other parameters, and put the animal head into the water with its head facing the barrel wall. The mice swim freely in the bucket to find a real platform, and the video system above the bucket captures their swimming tracks. In the first experiment, if the mouse found the solid platform within 120s and stayed on the platform for 2s, the mouse was quickly taken out, dried with a towel and put back into the breeding cage; if the mouse failed to find the solid platform within 120s, the small mouse The mouse was placed on the table for 15 seconds, then taken out and dried and returned to the cage. The water maze experiment was carried out for 3 days, once every morning and afternoon. 7-carbonyl dehydroepiandrosterone can enhance the spatial learning and memory ability of depressed mice (Figure 4).

8. Effect of 7-carbonyl dehydroepiandrosterone on Con A-stimulated proliferation of T lymphocytes in depressed mice

After the measurement of locomotor activity, the mice were sacrificed by decapitation. Prepare mouse spleen single-cell suspension according to routine, add cell suspension (2×10 ⁶ cells/ml) 100 μl and Con A 2 μg/ml to each well of a 96-well culture plate, and control wells with complete medium instead of Con A. Four replicate tubes were set up for both the experiment and the control. The culture plate was cultured at 37°C and 5% CO ₂ for 72 hours. 8 hours before the end of the culture, 37 kBq ³ H-TdR (specific radioactivity 0.93 TBq·mmol ^-1 ) was added to each well, and then the cells were harvested on a cell harvester. The filter membrane was dried, and 5ml of liquid scintillation liquid was added and placed on a liquid scintillation counter for measurement. 7-carbonyl dehydroepiandrosterone can enhance the proliferative ability of T lymphocytes stimulated by Con A in depressed mice (Fig. 5).

9. The effect of 7-carbonyl dehydroepiandrosterone on the killing activity of NK cells in depressed mice

Use the culture medium to adjust the cell density to 4×10 ⁶ ml ^-1 , take the mouse lymphoma YAC-1 cells as the target cells, add 75 μl of splenocyte suspension to each well of the 96-well culture plate, and press effector cells: target cells = Add 75 μl of YAC-1 target cell suspension at a ratio of 50:1, and set effector cells and target cells as controls. 150 μl per well, 4 parallel tubes were set up, cultured for 10 h at 37° C. and 5% CO ₂ , and the A value was determined by MTT method. 7-carbonyl dehydroepiandrosterone can enhance the killing activity of NK cells in depressed mice (Figure 6).

Claims (3)

1. The present invention relates to a synthetic compound 7-carbonylepiandrosterone capable of improving depression symptoms in chronic mild stress mice, characterized in that the molecular weight is 302.4 and the molecular formula is C ₁₉ H ₂₇ O ₃ . 2. 7-carbonylepiandrosterone according to claim 1 is characterized in that: it can significantly increase the amount of sucrose solution drinking in depressed mice, reverse the loss of euphoria; it can significantly increase the spontaneous activity of depressed mice, thereby improving the depression of mice symptom. Improve immune dysfunction and decline in learning and memory associated with depression in mice. 3. according to the described 7-carbonyl epiandrosterone of claim 1,2, it is characterized in that: can be used as new antidepressant candidate drug, and can be used as immunostimulant and learning and memory of hypoimmune function accompanying depression Accelerators for barriers.

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