CN111789846A - The application of levothyroxine and its pharmaceutically acceptable salts in the preparation of antidepressant drugs - Google Patents

Abstract

The invention discloses application of L-securinine and a medicinal salt thereof in preparing an anti-depression medicament. The antidepressant drug disclosed by the invention is single-component L-securinine and a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the L-securinine and the pharmaceutically acceptable salt thereof; the medicament or the pharmaceutical composition adopts the dosage forms of tablets, capsules, solutions, granules, powder, pills, sprays, suspensions, injections or instillations. The medicine or the medicine composition and the medicinal salt thereof can be applied to the treatment of acute depression and chronic depression and have the characteristics of obvious curative effect, quick response and small toxic and side effect.

Description

The application of levothyroxine and its pharmaceutically acceptable salts in the preparation of antidepressant drugs

technical field

The invention belongs to the field of medicine, and in particular relates to the application of levothyroxine and a pharmaceutically acceptable salt thereof in the preparation of antidepressant medicines.

Background technique

Depression is a common mental illness characterized by low mood, often accompanied by suicidal tendencies (FerrariA J, et al. PLoS Medicine , 2013, 10(11): e1001547). According to the data released by the World Health Organization, about 350 million people in the world suffer from depression, and about 1 million people with depression commit suicide every year, and there are more than 61 million people with depression in China. With the development of society and the accelerated pace of life, the number of patients is expected to continue to increase, bringing a heavy economic burden to the society (Phillips MR, et al, Lancet , 2009, 373(9680): 2041-2053). Currently, the first-line antidepressant drugs in clinical use are mainly selective serotonin reuptake inhibitors, such as fluoxetine, paroxetine and sertraline. Low levels of serotonin in the body. However, these drugs have two significant limitations: 1) slow onset of action, typically starting after 5–7 weeks of administration, and 2) low response, with only about one-third of patients responding to the drug. More notably, in some cases, the drug worked as an antidepressant without a detectable increase in serotonin. This evidence suggests that serotonin levels may not be directly linked to depression levels and may not be the most effective target for antidepressants.

Studies have shown that patients with depression have lesions in multiple regions of the brain, of which the most stable changes are neuronal loss, atrophy, decreased expression of dendritic proteins, and decreased number of dendritic spines and synapses in the hippocampus and prefrontal cortex (Krishnan Vand et al. Nestler EJ, Nature , 2008, 455(7215): 894; Kang HJ, et al, Nature Medicine , 2012, 18(9): 1413-1417). In animal models of depression, brain pathological features consistent with patients also appear, such as reduced branching, shortened length, reduced dendritic spines, and synaptic atrophy of hippocampal neurons (Duman RS, etal, Nature Medicine , 2016, 22(3): 238). As an important regulatory pathway in neurons, the mTOR molecular signaling pathway can improve the expression of synaptic proteins by regulating autophagy, transcription, translation and other processes, thereby promoting synaptic function, and many traditional antidepressants can also activate mTOR to a certain extent. Therefore, mTOR may become an important target of antidepressants (Ignácio ZM, et al, Br J Clin Pharmacol, 2016, 82(5):1280-1290). Ketamine, the first antidepressant that is different from serotonin reuptake inhibitors discovered in recent years, has the advantages of quick action and high response. Ketamine can adjust the function of neural networks and neuronal synapses by inhibiting the function of NMDA receptors and activating the mTOR pathway. Therefore, there is an urgent need to develop novel antidepressant drugs with high efficiency and low toxicity.

Securinine belongs to the indolizidine alkaloids, and the natural securinine includes levorotrine and dextrorotation. It has been found that L-monophylline has the effect of antagonizing the neurotransmitter receptor GABA _A receptor to excite the central nervous system (Beutler JA, et al, Brain Res , 1985, 3310 (1): 135-140), and has been clinically studied. The use of levorbitine nitrate injection is used to treat diseases such as facial nerve palsy, neurasthenia, narcolepsy and polio sequelae. However, the application of L-hagamine in depression has not been reported. The inventors found that levorbitine can increase the mTOR pathway level and synaptic protein level in the brain of depressed mice, and has significant curative effect on acute depression and chronic depression in mice, and is expected to be developed as a new drug for the treatment of depression.

SUMMARY OF THE INVENTION

The primary purpose of the present invention is to provide the application of L-monophylline or its pharmaceutically acceptable salts in the preparation of antidepressant drugs.

For realizing the above-mentioned purpose of the invention, the present invention adopts following technical scheme:

。A L-monophylline, having the chemical structure shown in formula (1):

.

The preparation method of above-mentioned levothyroxine and its pharmaceutically acceptable salt, comprises the following steps:

The raw material of Hagi Ichiba was dried and pulverized, extracted with 95% ethanol, and concentrated under reduced pressure to obtain the total extract of Ichiba Hagi extract; the total extract was suspended in water, and 10% aqueous hydrochloric acid was added to adjust the pH to 2-3. After chloroform extraction, the acid water layer was added with ammonia water to adjust the pH to 9-10, and then extracted with chloroform, and the chloroform layer was concentrated under reduced pressure to obtain the total alkaloids of Ichiba Hagi; the total alkaloids were purified by silica gel column chromatography to obtain L-Ichiba Hagi The alkali enriched part is recrystallized to obtain levorotatory hagine crystals.

Take levo-hagine crystals, slowly add dilute acid solution to dissolve the levo-hagine crystals completely, cool in an ice-water bath and then dry under reduced pressure to obtain a salt of levo-hagine, which is then dissolved in anhydrous ethanol and placed for re-use. Crystallize, filter and dry to obtain the pure product of levo-hagine pharmaceutically acceptable salt.

The above-mentioned pharmaceutically acceptable salts include but are not limited to: inorganic anions, such as chloride, bromide, iodide, sulfate, sulfite, nitrate, nitrite, phosphate and hydrogen phosphate, etc.; organic Anions such as acetate, propionate, cinnamate, benzyl sulfonate, citrate, lactate, and gluconate, among others.

L-Monophylline and its pharmaceutically acceptable salts can be formulated into a variety of dosage forms, including tablets, pills, dragees, granules, gels, ointments, solutions, suppositories, injections, inhalants, and sprays. These dosage forms can be used for both topical or systemic administration as well as immediate or sustained release administration. When administered by injection, the compounds can be formulated into solutions, suspensions and emulsions with water-soluble or fat-soluble solvents. When administered orally, it can be complexed with a pharmaceutically acceptable excipient using conventional techniques. These excipients can formulate the compounds into a variety of dosage forms acceptable to the patient, such as tablets, pills, capsules, suspensions, gels, and the like.

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

The invention provides new applications of levo-hagamine and its pharmaceutically acceptable salts in the treatment of depression. The experimental data show that L-monophylline has a significant effect on acute depression and chronic depression in mice, and it is effective after a single dose and has a rapid onset of action, and has no effect on the voluntary movement of animals. Therefore, the antidepressant drug prepared by levorotrine and its pharmaceutically acceptable salt has the characteristics of remarkable curative effect, quick onset of action and good safety.

Description of drawings

Figure 1: Pharmacokinetic curves of single intraperitoneal administration of L-monophylline into the blood and brain, where A is the plasma concentration of L-monophylline after intraperitoneal administration at different times, and B is L-monophylline Brain drug concentrations after intraperitoneal administration at different times.

Figure 2: L-monophylline reduces acute depression level in mice in forced swimming model: results of immobility time in forced experiment after intraperitoneal administration of L-monophylline for 30 min (A) or 90 min (B). 15 mice were tested in each group, the results are mean±SEM, and the statistical method is one-way ANOVA; *p<0.05, **p<0.01.

Figure 3: L-Monophylline activates AKT-mTOR-S6K, ERK and p38 pathways in the mouse brain: (A) 90 min after intraperitoneal injection of L-monophylline, the forebrain parts (mainly the cerebral cortex) were obtained. and hippocampus) after homogenization, Western blot experiment was performed to detect the response of each signaling pathway. (B-G) Statistical results of protein changes in different signaling pathways; the results are the mean±SEM of 6 groups of samples, and the statistical method is one-way ANOVA; *p<0.01, ***p<0.001.

Figure 4: Anti-acute depressive effect of oral administration of L-monophylline: statistical results of forced swimming immobility time after oral administration of monophylline for 3 hours. 15 mice in each group, the results are mean ± SEM, the statistical method is one-way ANOVA, *p<0.05.

Figure 5: Therapeutic effect of L-monophylline on chronic depression model mice: After administration of monophylline in chronic depression model mice, sugar water preference (A), forced swimming (B), and mTOR pathway activation (C) were tested , and PSD95 expression (D). It can be seen that a single dose can improve the sugar water preference disorder and increase the immobility time in forced swimming; the effect is more obvious after multiple doses. Regarding the activation of mTOR pathway and the expression of PSD95, the effect of single administration was not obvious, and the effect of multiple administration was significant. There are 15 mice in each group, the results are the mean±SEM, the statistical method is one-way ANOVA, **p<0.01, ***p<0.001, depression model+DMSO group vs normal mouse group; #p<0.05, # #p<0.01, ###p<0.001, depression model + levothyroxine group vs depression model + DMSO group; &&, depression model + single levomophylline group vs depression model + multiple times levothyroxine Hagiline group.

Figure 6: L-Hagine did not produce toxic side effects on the autonomous behavior of mice: After 60min administration of L-hagine, various indicators of mice's movement in the open field were tested: (A) The residence time in the central area; (B) Residence time in the peripheral area; (C) Movement distance in the central area; (D) Movement distance in the peripheral area; (E) Total movement distance; (F) Movement speed in the central area; (G) Peripheral area

Movement speed; (H) Total movement speed. There was no statistical difference in all indicators. There are 15 mice in each group, and the results are the mean of 15 mice in each group. The results are the mean ± SEM, and the statistical method is one-way ANOVA.

Detailed ways

The present invention will be described in further detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

Example 1: Separation and purification of L-monophylline

Take 1 kg of dried leaves and branches of Hagi, crush and extract with 5 L of 95% ethanol, extract 3 times, combine the extracts, and concentrate under reduced pressure to obtain 80 g of total extract; 50 mL of 10% hydrochloric acid aqueous solution was adjusted to pH 2~3, after extraction with chloroform, the acid water layer was adjusted to pH 9-10 by adding 30 mL of ammonia water, extracted with chloroform, and the chloroform layer was concentrated under reduced pressure to obtain Ichiba Hagi total alkaloids 0.9 g; the total alkaloids were purified by silica gel column chromatography, eluted with a chloroform-methanol mixed solvent, combined with the fractions rich in levothyroxine, and recrystallized to obtain about 200 mg of levoagarine crystals.

Embodiment 2: the preparation of levorotatory hagiline salt

100 mg of levothyroxine crystals (purity >95%), slowly add 6 mL of 1% hydrochloric acid solution to dissolve completely, place in an ice-water bath for 2 h, dry under reduced pressure and dissolve in 3 mL of anhydrous ethanol. Crystallization, the crystals were washed with methanol, filtered and dried to obtain L-monophylline hydrochloride (103 mg).

100 mg of levothyroxine crystals (purity >95%), slowly add 2 mL of 1% sulfuric acid solution to dissolve completely, place in an ice-water bath for 2 h, dry under reduced pressure and dissolve in 3 mL of anhydrous ethanol. Crystallization, the crystals were washed with methanol, filtered and dried to obtain L-monophylline sulfate (108 mg).

100 mg of levothyroxine crystals (purity > 95%), slowly add 3 mL of 1% nitric acid solution to dissolve it completely, place it in an ice-water bath for 2 h, dry it under reduced pressure and dissolve it with 3 mL of anhydrous ethanol. Crystallization, the crystals were washed with methanol, filtered and dried to obtain levorotatory hagiline nitrate (110 mg).

Example 3: Neural cell differentiation-promoting activity of levothyroxine and several salts thereof

Objective: To investigate the neuronal differentiation-promoting activity of L-monophylline and its nitrates, hydrochlorides and sulfates

Methods: Neuro-2a cells (purchased from American type culture collection cell bank) were recovered, cultured in growth medium (MEM+10%FBS+100 U/mL penicillin and 100 μg/mL streptomycin), and seeded at 100 mm in a petri dish and cultured in a constant temperature incubator containing 5% CO2 at 37°C. When the cells grow to 60–70%, the culture medium in the petri dish is aspirated, washed with an appropriate amount of PBS, and then digested with 0.25% trypsin for 45 seconds. Passage evenly at 1:10, every three days. When inducing neuronal cell line differentiation, the cell seeding density was 2×10 ⁴ cells/35 mm culture dish or 1×10 ⁴ cells/well (12-well plate), and the cells were cultured in growth medium for 24 hours and then changed to differentiation medium (MEM). + 0.5% FBS + 100 U/mL of penicillin and 100 μg/mL of streptomycin), and added L-monophylline or its several pharmaceutically acceptable salts for 48 hours. Cells were fixed with 4% paraformaldehyde/4% sucrose for 20-30 min, and the differentiated morphology and neurites of neural cell lines were observed by immunofluorescence staining with β-tubulin III (a marker protein specifically expressed in neurites). High-content instruments were used for automatic scanning and photography, and Cellomics view software was used for statistical analysis. Cells with a neurite length greater than twice the soma were defined as neurons, and statistical analysis was performed on the average length of total neurites per differentiated cell.

RESULTS: L-monophylline and its nitrate, hydrochloride, and sulfate salts all had similar neuronal differentiation-promoting activities (Table 1).

Table 1. Neural cell differentiation-promoting activity of L-monophylline and its salts

The values in table ^a are the neurite lengths (unit: μm), the results are the mean ± SEM of three independent experiments, and the statistical method is one-way ANOVA. ^b is the positive control, all-trans retinoic acid (RA).

Example 4: The synaptogenesis-promoting activity of L-monophylline and its several pharmaceutically acceptable salts

Objective: To investigate the synapse formation-promoting activity of L-hagine and its nitrates, hydrochlorides and sulfates

Methods: The hippocampus tissue of Sprague Dawley rat fetuses at 18 days of embryonic period was taken, and then single isolated neurons were obtained after digestion with trypsin. ) coated on 18 mm glass slides at a density of 0.2×10 ⁵ /slide, cultured in Neurobasal medium supplemented with 2% B27. The medium (Neurobasal medium with 2% B27) was changed every three days, half every time. On the 14th day of culture, DMSO or L-monophylline or several pharmaceutically acceptable salts (10 μM) were added and treated for 48 hours. Fix with 4% paraformaldehyde for 5 minutes and then with pre-cooled methanol for 15 minutes on ice. Afterwards, the synapse formation was evaluated by immunofluorescence staining with PSD-95 (marker protein specifically expressed in excitatory nerve postsynapses) antibody. Photographs were taken with a Zeiss Imager A2 fluorescence microscope and the number of PSD-95 clusters on average dendritic length in each group was counted.

Results: L-monophylline and its nitrates, hydrochlorides and sulfates all had similar activity in promoting PSD-95 clustering (Table 2).

Table 2. Activity of L-hagine and its salts in promoting PSD-95 clustering

Example 5: Pharmacokinetic results of blood and brain entry after intraperitoneal injection of levophylline

OBJECTIVE: To study the pharmacokinetic parameters of levothyroxine into the blood and brain

method:

Male C57BL/6J mice aged 7-8 months, fasted for 12 hours on the 1st day before the experiment, had free access to water, and were intraperitoneally injected with L-monophylline at a dose of 7.5 mg/kg. The preparation method was shown in Example 3. Before administration (0 min) and 1.5, 5, 9, 15, 30, 60, 120, 240, and 480 min after administration at 10 time points (3 mice at each time point), the eyeballs were removed and blood was collected. Take brain tissue. Blood samples were centrifuged (4°C, 3000 rpm, 8 min) to separate plasma.

Take 100 μL of mouse plasma, add 600 μL of acetonitrile (internal standard, IS) solution containing the internal standard compound, vortex for 5 min, centrifuge at 4 °C and 15000 rpm for 15 min, take the supernatant, and evaporate it with a vacuum concentrator. After drying (40 °C, 4 h), 100 μL of 50% methanol was added to reconstitute, vortexed for 3 min, sonicated for 5 min, vortexed for 1 min, and then centrifuged at 15,000 rpm for 15 min, and the supernatant was collected for LC/MS analysis.

After the brain tissue was washed with normal saline, the surface blood was stained, the water was absorbed, and weighed accurately. The ratio of mass (g) to volume (mL) was 1:2 with normal saline and homogenized. Take 200 μL of brain homogenate sample, add 1200 μL of acetonitrile solution containing internal standard compound respectively, vortex for 5 min, centrifuge at 4 °C and 15000 rpm for 15 min, take the supernatant and evaporate to dryness with a vacuum concentrator, 100 μL of 50% Reconstituted in methanol, processed as above and analyzed by LC/MS.

Chromatographic conditions: Waters ACQUITY QTOF quadrupole tandem time-of-flight mass spectrometry was used for effective separation of levorotyl hagiline and internal standard, the column temperature was set to 40 °C, the sample chamber temperature was 5 °C, and the mobile phase was pure water and acetonitrile, water Contains 0.1% formic acid. Chromatographic column: Waters ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 mm × 100 mm) reverse chromatographic column; time: 6 min; injection volume: 5 μL (see the table below for specific conditions).

Mass spectrometry conditions: positive ion scanning mode, electrospray ionization source, ion source temperature 60 °C; ion spray voltage 3000 V; scanning range: 100–500 Da; the transition [M+H+] of (+)-SE is 218.12. According to the peak areas of plasma and brain collected at each time point after administration of each test mouse, the plasma or brain concentration at each time point was calculated by the corresponding standard curve of blood or brain of each compound, and a non-compartmental model was used. The main pharmacokinetic parameters were calculated by the method, and parameters such as half-life were analyzed by WinNonlin version 6.3 (Pharsight, Mountain View, CA, USA) software.

RESULTS: L-Hagine could rapidly enter the blood and brain as the prototype, and the peak time was 9 minutes and 30 minutes, respectively, and the elimination time was 4 hours (Table 3 and Figure 1).

Table 3. Pharmacokinetic results of blood and brain entry after intraperitoneal injection of L-monophylline

Example 6: Therapeutic effect of L-monophylline on acute depressive episodes induced by forced swimming model

OBJECTIVE: To evaluate the anti-acute depression effect of L-monophylline

method:

1) C57BL/6J (6-7 weeks) male mice were housed in a 12-hour light (8:00 to 20:00), 12-hour dark cycle (20:00 to 8:00 the next day), and a relative humidity of 60–70 % of the animal room, free access to water and food. After one week of adaptive feeding (7-8 weeks), the experiment was started.

2) Mice were placed in the behavioral experimental room for more than 1 hour in advance.

3) Single intraperitoneal injection or oral administration: L-monophylline [(-)-SE] injection dose is 3.75 mg/kg or 7.5 mg/kg, oral dose is 10 mg/kg and 30 mg/kg. The preparation method of the medicine is as follows: completely dissolve the levothyroxine in DMSO, the concentrations are 37.5 mg/mL, 75 mg/mL, 100 mg/kg and 300 mg/kg respectively, take 1 volume of DMSO, add 1 volume of Tween-80 was thoroughly mixed, and then 98 volumes of normal saline was added and mixed thoroughly to obtain solutions with concentrations of 0.375 mg/mL, 0.75 mg/mL, 1 mg/kg, and 3 mg/kg, respectively. The volume for injection or oral administration 0.1 mL per 10 g of body weight. The blank group was given 1% DMSO, 1% Tween-80 and 98% normal saline as solvent control. Prepare freshly before each administration. There were 15 mice in each group. The forced swimming test was performed 30 min or 90 min after administration.

4) Turn on the video recorder (the angle is for side shooting), and place the mouse in a plexiglass tank with a diameter of 15 cm, a height of 25 cm, and a water depth of 13 cm (at this time, the hind paws of the mouse cannot reach the bottom), and the water temperature is (25 cm) ±1°C). The whole video was recorded for 6 minutes, and the immobility time of the mice in the last 4 minutes indicated the depression (despair) emotional level of the mice. After the experiment was completed, the mice were wiped dry with paper, and their brains were removed immediately after ether anesthesia and stored at -80°C for subsequent pathological experiments. Clean the glass jar and change the water before the next mouse experiment.

5) The mouse immobility time in the last 4 minutes in the video was analyzed by ForcedSwim Version 2.0 (Clever Sys Inc.) software.

RESULTS: L-monophylline showed antidepressant effects after a single intraperitoneal administration of 7.5 mg/kg for 30 min or 90 min, while 3.75 mg/kg also exhibited antidepressant effects 90 min after administration function (Figure 1). At the same time, the brain tissues of the depression group and the depression administration group were analyzed, and it was found that the mTOR pathway was significantly activated after administration, and the expression of the important functional protein PSD-95 in the synapse was also up-regulated, indicating that the synapse function was improved (Figure 2). ). In addition, oral administration at a dose of 30 mg/kg (administration time of 3 hours) also exhibited antidepressant effects (Figure 3).

Figure 2 shows that L-monophylline reduces the level of acute depression in mice in the forced swimming model: after intraperitoneal administration of L-monophylline for 30 min (A) or 90 min (B), immobility in the forced swimming experiment time result. 15 mice were tested in each group, results are mean ± SEM, statistical method is one-way ANOVA; *p < 0.05, **p < 0.01.

Figure 3 shows that L-monophylline activates AKT-mTOR-S6K, ERK and p38 pathways in the mouse brain, in which (A) 90 min after intraperitoneal injection of L-monophylline, the forebrain parts (mainly for The cerebral cortex and hippocampus) were homogenized and subjected to Western blot experiments to detect the response of each signaling pathway. (B-G) Statistical results of protein changes in different signaling pathways; the results are the mean ± SEM of 6 groups of samples, and the statistical method is one-way ANOVA; *p < 0.01, ***p < 0.001.

Figure 4 shows the anti-acute depressive effect of oral administration of L-monophylline: the statistical results of the immobility time of forced swimming 3 hours after oral administration of L-monophylline. 15 mice per group, results are mean ± SEM, statistical method is one-way ANOVA, *p < 0.05.

Example 7: Therapeutic effect of levorbitine on chronic depression model mice

OBJECTIVE: To evaluate the anti-chronic depression effect of L-monophylline

method:

1) C57BL/6J (6-7 weeks) male mice were housed in a 12-hour light (8:00 to 20:00), 12-hour dark cycle (20:00 to 8:00 the next day), and a relative humidity of 60–70 % of the animal room, free access to water and food. After one week of adaptive feeding (7–8 weeks), experiments were started.

2) The modeling method of chronic depression is as follows: Mice were sequentially given the stress modes shown in the following table, and there were two types of stress every day for 30 days. The mice were weighed every 5 days during the modeling period, and the depression level of the mice was detected by the sugar water preference test on the 15th and 30th days.

Stress Methods Used in Chronic Depression Modeling

3) Mice were divided into normal control group, depression model + DMSO solvent single-dose group, depression model + L-monophylline single-dose group, depression model + DMSO multiple-dose group, depression model + L-monophylline There were 15 rats in the phylloxine multiple-administration group. The single-administration group was administered the next day after the modeling, and the depression index was detected 90 minutes after the administration; the multiple-administration group was administered on the 16th day of modeling, once a day, for 15 days to the end of modeling ; Depression indicators were detected the next day. The administration methods are all intraperitoneal injection, and the dosage of levothyroxine is 7.5 mg/kg, and the preparation method is shown in Example 6.

4) The evaluation experiments of depression indicators include: forced swimming test, sugar water preference test, mTOR pathway, and detection of PSD-95 expression level.

Results: Both single- and multiple-dose groups exhibited antidepressant effects, including decreased immobility time in the forced swim test, increased sugar water intake in the sugar water preference test, decreased serum corticosterone levels, and increased mTOR pathway activation and PSD -95 expression level (Figure 5).

Figure 5 shows the therapeutic effect of L-monophylline on chronic depression model mice: after administration of L-monophylline in chronic depression model mice, sugar water preference (A), forced swimming (B), and serum corticosterone levels were tested (C), mTOR pathway activation (D), and PSD95 expression (E). Results are mean ± SEM, statistical method is one-way ANOVA, **p < 0.01, ***p < 0.001, depression model + DMSO group vs normal mouse group; #p < 0.05, ##p < 0.01, # ##p <0.001, Depression model + L-Monophylline group vs Depression model + DMSO group; &&, Depression model + Single L-Monophylline group vs Depression model + Multiple times L-Monophylline group.

Example 8: L-monophylline did not produce toxic side effects on the voluntary motor behavior of mice

Objective: To evaluate the toxic and side effects of levo-hagine

method:

C57BL/6J (7–8 weeks) male mice were acclimated to the experimental room (temperature 23–25°C) for 1 hour, and then intraperitoneally administered L-monophylline at 3.75 mg/kg or 7.5 mg/kg. The drug preparation method is as follows: as described in Example 6. After 30 minutes of single administration, an open field test was used to evaluate the autonomous behavior of the mice. The steps are as follows:

1) The open field is a 40 × 40 × 40 cm opaque polypropylene material (Polypropylene) experimental box with an opening on the top. The mice are taken out of the cage and placed in the center of the box. The top shooting starts to time, and the video system is used to record for 30 min;

2) Use TopScan Version 3.0 (Clever Sys Inc.) software to analyze the mouse movement trajectory, speed, residence time in the central area (50% of the area) and surrounding areas, and perform data statistics.

RESULTS: After administration of L-monophylline (3.75 mg/kg or 7.5 mg/kg), the movement distance, speed, and residence time in the center and four weeks of mice were all normal, indicating that these two doses have no effect on the autonomy of mice. Motor behavior had no toxic side effects (Figure 6).

Figure 6 shows that L-monophylline did not produce toxic side effects on the voluntary motor behavior of mice: 30 min after administration of L-monophylline, various indicators of mice's movement in the open field were tested: (A) stay in the central area time; (B) residence time in the outer peripheral area; (C) movement distance in the central area; (D) movement distance in the outer peripheral area; (E) total movement distance; (F) movement speed in the central area; (G) movement speed in the outer peripheral area; ( h) Total movement speed. There was no statistical difference in all indicators. There are 15 mice in each group, the results are the mean of 15 mice in each group, the results are the mean ± SEM, and the statistical method is one-way ANOVA.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

Claims (5)

1. The application of levorbitine and its pharmaceutically acceptable salts in the preparation of antidepressant drugs. 2 . The levothyroxine and pharmaceutically acceptable salts thereof according to claim 1 , wherein the pharmaceutically acceptable salts include hydrochloride, sulfate, nitrate and the like. 3 . 3. The application of levo-phyllophylline and pharmaceutically acceptable salts thereof in the preparation of antidepressant according to claim 1-2, characterized in that, the antidepressant is levorbitine and medicament thereof Use salts or pharmaceutical compositions containing the above ingredients. 4. according to the application of levothyroxine according to claim 1-3 and its pharmaceutically acceptable salt in the preparation of antidepressant, it is characterized in that, described antidepressant or pharmaceutical composition can be used in medically acceptable. Accepted modes of administration and dosage forms. 5. the mode of administration and the dosage form that levophylline according to claim 4 and its pharmaceutically acceptable salt or the pharmaceutical composition containing above composition are adopted in the preparation of antidepressant medicine, it is characterized in that, can pass the tablet It can be realized through preparations, capsules, solutions, granules, powders, pills, sprays, suspensions, injections or drips, etc.

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