Preparation and application of spasmodic drug with synergistic anti-depression effect and composition of spasmodic drug

Abstract

The invention discloses preparation and application of a spasm drug with a synergistic anti-depression effect and a composition of the spasm drug. Belongs to the field of medicine, and is desmethylvenlafaxine-gamma-aminobutyric acid as shown in a formula I; the pharmaceutical composition comprises a pharmaceutical composition of desmethylvenlafaxine and gamma-aminobutyric acid, and a pharmaceutical composition of venlafaxine and gamma-aminobutyric acid. The method further comprises the step of using the desmethylvenlafaxine-gamma-aminobutyric acid twin drug, a pharmaceutical composition of desmethylvenlafaxine and gamma-aminobutyric acid and a pharmaceutical composition of venlafaxine and gamma-aminobutyric acid to treat depression. The invention relates to application of a spasmodic drug or pharmaceutically acceptable salt and composition thereof or a composition of the spasmodic drug in preparation of drugs for preventing or treating depression. The desmethylvenlafaxine-gamma-aminobutyric acid twin drug, the pharmaceutical composition of desmethylvenlafaxine and gamma-aminobutyric acid and the pharmaceutical composition of venlafaxine and gamma-aminobutyric acid are used for preventing or treating depression, especially for depression accompanied by anxiety, and the drug or the composition can improve the curative effect of the drug and has better anti-anxiety and anti-depression effects. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of medicine, and relates to the preparation and application of a type of anticonvulsant medicine with synergistic antidepressant effect and a composition thereof. Background Art

[0002] Depression is a common mental disorder involving prolonged low mood or loss of pleasure or interest in activities. Depression is one of the most common mental illnesses in the world. Depression already accounts for 10% of the total global non-fatal disease burden and is expected to become the leading cause of global disease burden by 2030.

[0003] Venlafaxine (VLF) is a phenylethylamine derivative and a second-generation antidepressant chiral drug. Studies have shown that venlafaxine mainly inhibits the reuptake of 5-HT when treated with low doses, and inhibits the reuptake of 5-HT and NE when treated with high doses. Its structure is shown in Formula II. Its main active metabolite is O-desmethylvenlafaxine (ODV), and its structure is shown in Formula III. Venlafaxine and desmethylvenlafaxine are suitable for various types of depression, including depression with anxiety and generalized anxiety disorder, but the drug has the disadvantage of limited efficacy in treating depression and anxiety.

[0004] Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a class of anticonvulsant drugs with synergistic antidepressant effects and the preparation and application of their compositions.

[0006] Technical solution: The spasticity drug described in the present invention is a compound formed by condensation connection of desvenlafaxine and γ-aminobutyric acid or a pharmaceutically acceptable salt or a pharmaceutically acceptable carrier thereof, and its chemical structure is shown in Formula I.

[0007]

[0008] A composition comprising desvenlafaxine and gamma-aminobutyric acid, or venlafaxine and gamma-aminobutyric acid.

[0009] A composition of a spastic medicine comprises the compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0010] A method for preparing a type of spasticity drug with synergistic antidepressant effect: the method comprises the following steps:

[0011] Step 1: Protect the amino group in the γ-aminobutyric acid structure with di-tert-butyl dicarbonate or benzyl chloroformate to obtain intermediate I;

[0012] Step 2: subjecting desvenlafaxine to a condensation reaction with the intermediate I obtained in the first step under EDCI / DMAP to obtain intermediate II;

[0013] Step 3: Remove the protecting group of the intermediate II obtained in the second step under acidic or palladium-carbon conditions to obtain the spasm drug; the synthetic route is as follows:

[0014]

[0015] Further, the weight ratio of desvenlafaxine to γ-aminobutyric acid is 0.1-5:1;

[0016] The weight ratio of venlafaxine to gamma-aminobutyric acid is 0.1-5:1.

[0017] Furthermore, the dosage of desvenlafaxine or a pharmaceutically acceptable salt thereof is 10 to 375 mg of desvenlafaxine, the dosage of γ-aminobutyric acid or a pharmaceutically acceptable salt thereof is 2 to 100 mg of γ-aminobutyric acid base, and the dosage of venlafaxine or a pharmaceutically acceptable salt thereof is 10 to 225 mg of venlafaxine base.

[0018] Furthermore, a desvenlafaxine-γ-aminobutyric acid anesthetic or a composition thereof, a composition of desvenlafaxine and γ-aminobutyric acid, a composition of venlafaxine and γ-aminobutyric acid, and a use of the compound or a pharmaceutically acceptable salt thereof, the composition or the combination of the anesthetic in the preparation of a drug for preventing or treating depression.

[0019] Furthermore, the depression is major depression, bipolar depression, psychotic depression, reactive depression, menopausal depression, childhood depression, secondary depression, postpartum depression, seasonal depression, persistent depression, depression in postmenopausal and perimenopausal women, and anxiety depression.

[0020] According to in vivo microdialysis studies, venlafaxine and desvenlafaxine can quickly penetrate the brain and increase the concentration of norepinephrine in the hypothalamus, but not dopamine concentration, so they have a highly selective effect on serotonin and norepinephrine monoamine transporters. Therefore, the antidepressant mechanism of venlafaxine and desvenlafaxine is to inhibit the transporters of serotonin and norepinephrine in the presynaptic membrane, inhibit the reuptake of 5-HT and NE, increase the concentration of neurotransmitters in the synaptic cleft, thereby enhancing the signal transduction of the postsynaptic membrane and activating the postsynaptic receptors involved in the regulation of emotional states. Venlafaxine and desvenlafaxine are now used to treat major depressive disorder, persistent depressive disorder, childhood depression, and depression in postmenopausal and perimenopausal women. However, the drug has disadvantages such as a long onset period and limited relief of anxiety symptoms.

[0021] The chemical name of γ-aminobutyric acid (GABA) is 4-aminobutyric acid, and its structure is shown in Formula IV. It is a non-proteinogenic amino acid that is widely found in microorganisms, plants, and vertebrates. γ-aminobutyric acid is the main inhibitory neurotransmitter in the brain, and maintains homeostasis by balancing the excessive excitability of neurons associated with diseases such as epileptic seizures and anxiety. After the γ-aminobutyric acid A receptor is activated, it can selectively allow chloride ions to pass through, increase the opening frequency of chloride ion channels, cause neuronal hyperpolarization, reduce the membrane excitability of nerves or other tissues, and then inhibit signal transmission. γ-aminobutyric acid energy transmission occurs in interneurons, and interneurons can regulate local neurotransmission, including noradrenergic, dopaminergic, and serotonergic neurons. Therefore, its physiological effects are related to regulating synaptic transmission, promoting neuronal development and relaxation, preventing insomnia, and improving anxiety and depression. A large amount of information supports the role of the GABA system in the pathophysiology of anxiety. Studies have found that the concentration of GABA in the brains of patients with depression is reduced. At the same time, animal experiments have also shown that enhancing the activity of GABA neurons and increasing the level of GABA neurotransmitters have antidepressant effects in depression models.

[0022]

[0023] Twin drugs refer to new multifunctional molecules formed by combining and connecting two identical or different lead compounds or drugs through covalent bonds, which produce synergistic effects in the body, enhance activity or produce new pharmacological activities, or improve the selectivity of the action. The two molecules combined into twin drugs can have the same type of pharmacological action. For example, both aspirin and paracetamol have antipyretic and analgesic activity. The new molecule Benorilate is obtained by esterification of the two, which has a synergistic effect, which not only solves the acidic irritation of aspirin to the stomach, but also enhances the efficacy. In addition, two drugs with different pharmacological effects can also be combined to form twin drugs to produce new or combined effects. For example, chlorambucil is an antitumor drug, but it is highly toxic. Designing with steroids as carriers can increase targeting. This idea is used to form the antitumor drug prednimustine with prednisolone and chlorambucil, which reduces the toxicity of chlorambucil.

[0024] The beneficial effects of the present invention are as follows: the desvenlafaxine γ-aminobutyric acid sedative, the desvenlafaxine and γ-aminobutyric acid composition, and the venlafaxine and γ-aminobutyric acid composition of the present invention have synergistic antidepressant and antianxiety effects in the treatment of depression. The effective dose of a single component can be effectively reduced through different antidepressant mechanisms; the present invention verifies the synergistic antidepressant and antianxiety effects between the above-mentioned drugs by the equimolar method, and has no obvious toxic and side effects while exerting the antidepressant and antianxiety effects, and has a good clinical application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the hydrogen spectrum of 4-((tert-butoxycarbonyl)amino)butanoic acid (intermediate I) in the embodiment of the present invention;

[0026] Figure 2 is the hydrogen spectrum of 4-(2-(dimethylamino)-1-(1-hydroxycyclohexyl)ethyl)phenyl 4-((ter t-butoxycarbonyl)amino)butanoate (Intermediate II) in the embodiment of the present invention;

[0027] Figure 3 is the hydrogen spectrum of 4-(2-(dimethylamino)-1-(1-hydroxycyclohexyl)ethyl)phenyl 4-ami nobutanoate (desmethylvenlafaxine-γ-aminobutyric acid) in the embodiment of the present invention;

[0028] Figure 4 Schematic diagram of the effect of desvenlafaxine-γ-aminobutyric acid on the behavior of mice in the chronic unpredictable mild stress model according to the embodiment of the present invention ( Figure 4 A: 28-day weight change; Figure 4 B: 12h sucrose preference experiment; Figure 4 C: tail suspension test after 4 min; Figure 4 D: forced swimming test after 4 min; Figure 4 E: Total distance moved by mice in 5 min in the open field test; Figure 4 F: 5-min movement trajectory of mice in the open field test; Figure 4 G: Total movement time of mice in the open field test for 5 min; Figure 4 H: 5-min center zone time of mice in the open field test);

[0029] Figure 5 Schematic diagram of the effect of desvenlafaxine-γ-aminobutyric acid on neurotransmitters in the brain of mice in a chronic unpredictable mild stress model according to an embodiment of the present invention ( Figure 5A: 5-HT content in mouse hippocampus; Figure 5 B: Norepinephrine content in mouse hippocampus; Figure 5 C: GABA content in mouse hippocampus);

[0030] Figure 6 Schematic diagram of the effect of desvenlafaxine-γ-aminobutyric acid on the synaptic structural plasticity of neurons in the hippocampal CA1 region of mice in the chronic unpredictable mild stress model according to the present invention ( Figure 6 A: Representative images of dendritic spine density in the CA1 region of mouse hippocampus; Figure 6 B: Dendritic spine density analysis in the CA1 region of the mouse hippocampus; Figure 6 C: Neuronal branch path tracing diagram in the CA1 region of mouse hippocampus; Figure 6 D: Analysis of dendritic complexity of neurons in the CA1 region of mouse hippocampus);

[0031] Figure 7 Schematic diagram of the effect of the desvenlafaxine and γ-aminobutyric acid composition on the behavior of mice in the chronic unpredictable mild stress model according to the embodiment of the present invention ( Figure 7 A: 28-day weight change; Figure 7 B: 12h sucrose preference experiment; Figure 7 C: tail suspension test after 4 min; Figure 7 D: forced swimming test after 4 min; Figure 7 E: Total distance moved by mice in 5 min in the open field test; Figure 7 F: 5-min movement trajectory of mice in the open field test; Figure 7 G: Total movement time of mice in the open field test for 5 min; Figure 7 H: 5-min center zone time of mice in the open field test);

[0032] Figure 8 Schematic diagram of the effect of the desvenlafaxine and γ-aminobutyric acid composition on neurotransmitters in the brain of mice with chronic unpredictable mild stress model according to the embodiment of the present invention ( Figure 8 A: 5-HT content in mouse hippocampus; Figure 8 B: Norepinephrine content in mouse hippocampus; Figure 8 C: GABA content in mouse hippocampus);

[0033] Fig. 9 Schematic diagram of the effect of the desvenlafaxine and γ-aminobutyric acid composition on the synaptic structural plasticity of neurons in the hippocampal CA1 region of the chronic unpredictable mild stress model mice in the present invention ( Fig. 9 A: Representative images of dendritic spine density in the CA1 region of mouse hippocampus; Fig. 9 B: Dendritic spine density analysis in the CA1 region of the mouse hippocampus; Fig. 9C: Neuronal branch path tracing diagram in the CA1 region of mouse hippocampus; Fig. 9 D: Analysis of dendritic complexity of neurons in the CA1 region of mouse hippocampus);

[0034] Fig.10 Schematic diagram of the effect of the venlafaxine and γ-aminobutyric acid composition on the behavior of mice in the chronic unpredictable mild stress model according to the embodiment of the present invention ( Fig.10 A: 28-day weight change; Fig.10 B: 12h sucrose preference experiment; Fig.10 C: tail suspension test after 4 min; Fig.10 D: forced swimming test after 4 min; Fig.10 E: Total distance moved by mice in 5 min in the open field test; Fig.10 F: 5-min movement trajectory of mice in the open field test; Fig.10 G: Total movement time of mice in the open field test for 5 min; Fig.10 H: 5-min center zone time of mice in the open field test);

[0035] Fig.11 Schematic diagram of the effect of the venlafaxine and γ-aminobutyric acid composition on neurotransmitters in the brain of mice with chronic unpredictable mild stress model according to the embodiment of the present invention ( Fig.11 A: 5-HT content in mouse hippocampus; Fig.11 B: Norepinephrine content in mouse hippocampus; Fig.11 C: GABA content in mouse hippocampus);

[0036] Fig.12 Schematic diagram of the effect of the venlafaxine and γ-aminobutyric acid combination on the synaptic structural plasticity of neurons in the hippocampal CA1 region of mice in the chronic unpredictable mild stress model according to the present invention ( Fig.12 A: Representative images of dendritic spine density in the CA1 region of mouse hippocampus; Fig.12 B: Dendritic spine density analysis in the CA1 region of the mouse hippocampus; Fig.12 C: Neuronal branch path tracing diagram in the CA1 region of mouse hippocampus; Fig.12 D: Analysis of dendritic complexity of neurons in the CA1 region of the mouse hippocampus). DETAILED DESCRIPTION

[0037] In some embodiments, the spasticity medicine can be used in combination with at least one other therapeutic drug, and the other therapeutic drug is selected from anti-schizophrenia drugs, antianxiety drugs, antidepressants, anti-epileptic drugs, and anti-Alzheimer's drugs. The other therapeutic drugs can be administered before, at the same time, or after the spasticity medicine of the present invention is used.

[0038] The desvenlafaxine-γ-aminobutyric acid sedative or composition of the present invention and pharmaceutically acceptable excipients are prepared in a form suitable for administration by any appropriate route, and the active sedative is preferably in a unit dose form, or in a form in which a patient can self-administer a single dose. The unit dose of the sedative or composition provided by the present invention can be expressed in the form of tablets, capsules, injections, granules, tinctures, lozenges, suppositories, regeneration powders or liquid preparations.

[0039] The dosage of the anticonvulsant drug or composition used in the method of use generally varies with the severity of the disease, the patient's weight and the relative efficacy of the compound. As a general guide, a suitable unit dosage may be 0.01 to 1000 mg.

[0040] The present invention also provides a method for treating depression, the method comprising administering the compounds of this invention to a subject in need of a therapeutically effective amount. In the present invention, the treatment of depression includes but is not limited to: antidepressant-related symptoms, improving depression-related symptoms, treating depression-related symptoms, preventing depression-related symptoms. In some embodiments, compared with administering a single portion alone, administering a spasm medicine or pharmaceutical composition of the present invention can improve therapeutic activity. In certain embodiments, spasm medicine or pharmaceutical composition are administered in combination with other drugs. The other drugs can be administered before, simultaneously or afterwards administering the compounds of this invention or pharmaceutical composition.

[0041] Example

[0042] Example 1. Synthesis of desvenlafaxine-γ-aminobutyric acid (ODV-GABA)

[0043] Reaction:

[0044]

[0045] The intermediate I is obtained by reacting γ-aminobutyric acid with di-tert-butyl dicarbonate, and further reacted with desvenlafaxine to generate intermediate II, and finally the target product desvenlafaxine-γ-aminobutyric acid is obtained under acidic conditions;

[0046] First, 5.0 g (48.48 mmol, 1.0 eq) of γ-aminobutyric acid and 40 mL of water were added to a 100 mL round-bottom flask. 3.87 g (96.75 mmol, 2.0 eq) of sodium hydroxide was slowly added with stirring, and 12.69 g (58.14 mmol, 1.2 eq) of di-tert-butyl dicarbonate was dropwise added. After the addition was completed, the mixture was reacted at room temperature for 4 h. The reaction was monitored by TLC and the reaction was complete. After 15 mL × 3 of dichloromethane was added for extraction, the pH was adjusted to about 3 to 4 with 1 mol / L hydrochloric acid solution, and then extracted with 15 mL × 3 of dichloromethane. The organic phase was dried and concentrated to obtain 8.67 g of white oil of intermediate I with a yield of 88.29%.1 H NMR(500MHz,Chloroform-d)δ4.78(s,1H),3.77–3.70(m,1H),3.19(t,J=7.0Hz,2H),2.43–2.34(m,2H),1.83(m,J=7.0Hz,2H),1.45(s,9H).(-)-HR-ESI-MS m / z 203.11576(calcd.202.11522for C 9 H 16 NO 4 - [MH] - );

[0047] Secondly, 5 g (24.6 mmol, 1.3 eq) of intermediate I, 5 g (18.98 mmol, 1.0 eq) of desvenlafaxine, 0.23 g (18.82 mmol, 0.1 eq) of DMAP, 4.36 g (22.74 mmol, 1.2 eq) of EDCI, and 50 mL of dichloromethane were added to a 100 mL round-bottom flask. After reacting at room temperature for 3 h, the reaction was monitored by TLC. The reaction was basically completed, 50 mL of water was added, and the mixture was dried and concentrated after extraction; organic phase column chromatography (dichloromethane: methanol = 50: 1) was performed to obtain 6.21 g of intermediate II as a white solid with a yield of 64.59%. 1 H NMR(500MHz,Chloroform-d)δ7.12(d,J=8.6Hz,2H),6.99(d,J=8.6Hz,2H),4.67(s,1 H),3.27(q,J=14.6,13.5Hz,3H),2.99(m,J=12.4,3.3Hz,1H),2.60(t,J=7.3Hz,2H),2 .31(s,6H),2.29(d,J=3.3Hz,1H),1.92(m,J=7.0Hz,2H),1.76–1.60(m,3H),1.53(q, J=13.9Hz,3H),1.44(s,9H),1.40–1.22(m,3H),0.98–0.81(m,2H).(+)-HR-ESI-MSm / z 448.29372(calcd.449.29451for C 25 H 41 N 2 O 5 + [M+H] + )

[0048] Finally, 0.35 g of intermediate II was added to a 25 mL round-bottom flask, 5 mL of 2M HCl / EA solution was added dropwise, and the reaction was protected by nitrogen at room temperature; after 2 h, the reaction was complete as monitored by TLC; 5 mL of water was added, and the pH was adjusted to about 9 to 10 with a saturated sodium bicarbonate aqueous solution, and extracted with 8 mL of dichloromethane × 3 times. After the organic phases were combined, dried and concentrated, column chromatography (dichloromethane: methanol = 45:1) was performed to obtain 0.2 g of desvenlafaxine-γ-aminobutyric acid as a white solid, with a yield of 73%. 1 H NMR(500MHz,DMSO-d6)δ7.40(d,J=8.1Hz,2H),7.12(d,J=8.0Hz,2H),3.66(s,1H),3.53 (s,1H),3.16(s,2H),2.86(q,J=6.7Hz,2H),2.73(t,J=7.4Hz,2H),2.68(d,J=4.6Hz,3H) ,2.61(d,J=4.5Hz,3H),1.93(t,J=7.6Hz,2H),1.65(d,J=13.2Hz,1H),1.56(d,J=11.4H z,1H),1.43(d,J=8.9Hz,3H),1.34(s,1H),1.27–1.13(m,4H),1.10–0.92(m,2H).(+)-HR ESI-MS: m / z349.2492(calcd.349.2486for C 20 H 33 N 2 O 3 + [M+H] + )

[0049] Example 2: Synthesis of Desvenlafaxine-γ-Aminobutyric Acid (ODV-GABA)

[0050] 1 g of intermediate II and 5 mL of dichloromethane were placed in a 50 mL round-bottom flask. 3 mL of trifluoroacetic acid was added dropwise at 0°C. After 1 h, the reaction was completed as monitored by TLC. Desvenlafaxine-γ-aminobutyric acid trifluoroacetate was obtained by column chromatography (dichloromethane:methanol=40:1) as a white solid (0.58 g). Yield: 56.31%.

[0051] Example 3: Synthesis of Desvenlafaxine-γ-Aminobutyric Acid (ODV-GABA)

[0052] In a 50 mL round-bottom flask, 1 g of intermediate II, 10 mL of toluene and 5 g of silica gel were placed, and the mixture was refluxed at 110° C. in an oil bath. After 4 h, the mixture was filtered off with suction, rinsed with ethyl acetate, and the organic phase was dried and concentrated. 0.18 g of desvenlafaxine-γ-aminobutyric acid as a white solid was obtained by column chromatography (dichloromethane:methanol=30:1). The yield was 23.38%.

[0053] Example 4. Synthesis of Desmethylvenlafaxine-γ-aminobutyric Acid (ODV-GABA)

[0054] In a 50 mL round-bottom flask, 1 g (2.22 mmol, 1 eq) of intermediate II and 10 mL of ethyl acetate were added dropwise. 0.29 g (2.3 mmol, 1.05 eq) of oxalic acid in 10 mL of ethyl acetate solution was added dropwise. After 2 h, the mixture was filtered and the filter cake was washed with ethyl acetate. After drying at 50 ° C in vacuum for 5 h, desvenlafaxine-γ-aminobutyric acid oxalate was obtained as a white solid of 1.2 g with a yield of 81.67%.

[0055] Example 5. Synthesis of Desvenlafaxine-γ-Aminobutyric Acid (ODV-GABA)

[0056] Reaction:

[0057]

[0058] First, 5 g (48.48 mmol, 1 eq) of γ-aminobutyric acid and 50 mL of water were added into a 250 mL round-bottom flask, and 5.13 g (48.4 mmol, 1 eq) of sodium carbonate was slowly added at 0°C and stirred; 30 mL of a 1,4-dioxane solution of 9.92 g (58.15 mmol, 1.2 eq) of CbzCl was slowly added dropwise; after the addition was completed, the reaction was stirred at room temperature for 3 h, and the reaction was basically completed after TLC monitoring; 50 mL of ethyl acetate was added for extraction, and the pH of the aqueous phase was adjusted to about 2 to 3 with 1 mol / L hydrochloric acid solution. After extraction with 50 mL of ethyl acetate for 3 times, the organic phase was dried and concentrated to obtain 6.55 g of a white oily intermediate III with a yield of 57%.

[0059] Then, 5 g (24.6 mmol, 1.3 eq) of intermediate III, 5 g (18.98 mmol, 1.0 eq) of desvenlafaxine, 0.23 g (18.82 mmol, 0.1 eq) of DMAP, 4.36 g (22.74 mmol, 1.2 eq) of EDCI, and 50 mL of dichloromethane were added to a 100 mL round-bottom flask. After reacting at room temperature for 3 h, the reaction was monitored by TLC, and the reaction was basically completed. 50 mL of water was added, and the organic phase was dried and concentrated after extraction, and column chromatography (dichloromethane: methanol = 50: 1) was performed to obtain 6.21 g of intermediate IV as a white solid with a yield of 64.42%.

[0060] Finally, 1 g (2.07 mmol, 1 eq) of intermediate IV, 0.02 g of Pd / C 10%, 0.39 g of ammonium formate (6.18 mmol, 3 eq), and 15 mL of methanol were added into a 50 mL round-bottom flask, and the mixture was refluxed at 80° C. for 4 h. The reaction solution was filtered, 10 mL of water was added to the filtrate, and the organic phase was dried and concentrated after extraction with 20 mL of dichloromethane × 2. Column chromatography (dichloromethane: methanol = 30:1) was performed to obtain 0.13 g of desvenlafaxine-γ-aminobutyric acid with a yield of 34.46%.

[0061] Example 6: Whole Blood Metabolic Stability Test of Desvenlafaxine-γ-Aminobutyric Acid

[0062] SPF male SD rats, weighing 180-300 g, were purchased from a certain city's Oriental breeding farm (experimental animal quality certificate number: NO.202370789); all animal facilities and feeding methods were in line with national standards; the laboratory temperature was 21±3°C, the humidity was 50±20%, and the rats were fed with 12h alternating light and dark; they were fasted for 12h before blood collection but could drink water; chromatographic column: AQ-C18, (2.1×50mm, 5μm); mobile phase: A: 0.1% formic acid-water; B: 0.1% formic acid-acetonitrile, injection volume was 10μl

[0063] The experimental process is as follows:

[0064] (1) Take an appropriate amount of blank plasma in an EP tube, add 100 μM working solution to a final concentration of 1 μM, and vortex to mix;

[0065] (2) Place the sample in a 37°C water bath in duplicate; incubate for 0, 5, 15, 30, and 60 min;

[0066] (3) Compounds: Take out 20 μl of sample from the incubation system at a certain time point, add 300 μl of internal standard precipitant, vortex mix, centrifuge the sample for 10 min (5500 g), take 150 μl of supernatant and add 150 μl of ultrapure water into a 96-well plate;

[0067] (4) Control: Take out 20 μl of sample from the incubation system at a certain time point, add 300 μl of internal standard precipitant, vortex mix, centrifuge the sample for 10 min (5500 g), take 150 μl of supernatant and add 150 μl of ultrapure water into a 96-well plate;

[0068] (5) Absolute zero point (compound): 18 μl of blank plasma was taken out, 300 μl of internal standard precipitant was added, 2 μl of 10 μM analyte working solution was added, vortexed and mixed, the sample was centrifuged for 10 min (5500 g), and 150 μl of supernatant was taken and added with 150 μl of ultrapure water in a 96-well plate;

[0069] (6) Vortex mix and inject for LC-MS / MS analysis.

[0070] Table 1 shows the metabolic half-life (t1 / 2) of propantheline, desvenlafaxine-γ-aminobutyric acid and desvenlafaxine-γ-aminobutyric acid-Boc in rat whole blood; metabolic half-life is an indicator of the metabolic rate of a drug in the body, and the lasting efficacy of desvenlafaxine is closely related to metabolism.

[0071] Table 1 Metabolic time of desvenlafaxine and desvenlafaxine-γ-aminobutyric acid in rat whole blood

[0072]

[0073] Pharmacological Examples

[0074] Establishment of the chronic unpredictable mild stress model (CUMS)

[0075] The chronic unpredictable mild stress model simulates the pathogenesis of human depression, studies the pathogenesis of depression, and provides an experimental basis for the screening of antidepressant drugs and the evaluation of their efficacy. This model is believed to be able to effectively induce depressive-like symptoms. The behavioral characteristics of animals will change after stress, and the model is long-lasting and stable. As a depression model, it has high value and is of great significance for the screening of depression drugs and the study of depression mechanisms.

[0076] In this experiment, the experimental method of modeling and drug administration was used to model the mice with chronic unpredictable mild stress and drug administration for 28 days. Except for the control group, each group of mice started modeling after drug administration every day. The mice in the normal control group were placed in a room with suitable temperature, humidity and light conditions. Except for fasting and water deprivation for 12 hours before sucrose preference, no stimulation was given. The mice in the other modeling groups received stress stimulation for 4 consecutive weeks. A total of 11 stressors were selected, including tail clamping (2-5 minutes), cold water swimming (12℃, 6-10 minutes), noisy sounds (5-12 hours), crowding (2-5 hours), restraint (0.5-2 hours), cage tilt (45°, 5-12 hours), no bedding ( The duration and intensity of different stressors were different to maximize the unpredictability of the scheme. The stressors started from 9:00 to 21:00 a.m., 2-3 stressors were randomly applied every day, and mice were allowed at least 2 hours of rest time between two stressors. The analysis of the chronic unpredictable mild stress model was mainly carried out through behavioral evaluation. Commonly used evaluation methods include sucrose preference test, tail suspension test, forced swimming test, open field test, etc., and secondly, it can be evaluated by neurotransmitter content and neuronal damage.

[0077] Evaluation of antidepressant model

[0078] Antidepressant Model Evaluation 1:

[0079] Body weight: The mice in each group were weighed on days 0, 7, 14, 21, and 28 of the experiment;

[0080] Antidepressant Model Evaluation II:

[0081] Sugar water preference test (SPT): After continuous modeling and drug administration for 4 weeks, the sugar water preference of mice was tested. All mice were housed in single cages. Before the test, each mouse was given a bottle of 1% sucrose solution and a bottle of pure water for 48 hours of sugar water training. Then the mice were fasted and deprived of water for 24 hours. Finally, a 12-hour test was conducted. The positions of the sugar water bottle and the pure water bottle were exchanged in the middle, and the sucrose consumption and pure water consumption within 12 hours were recorded; Sugar water preference index = sucrose drinking amount / (sucrose drinking amount + water drinking amount)*100%;

[0082] Antidepressant Model Evaluation III:

[0083] Tail suspension test (TST): The test was conducted after 4 weeks of continuous administration. First, the behavioral program was opened, and the background was selected to be in sharp contrast with the mouse's fur color. White mice were treated with a black background. Second, the mouse's tail was fixed with tape at the posterior 1 / 3 and hung on a bracket, with the head about 30 cm away from the table. Any-Maze software began to record the state of the mouse during the 6-min suspension. The first 2 minutes were the adaptation stage, and the last 4 minutes of immobility time of each mouse were analyzed. During the experiment, the environment was kept quiet to avoid external interference.

[0084] Antidepressant Model Evaluation IV:

[0085] Forced swimming test (FST): The test was conducted after 4 weeks of continuous administration. The mice were placed in a transparent glass container with a height of 28 cm and a diameter of 15 cm and filled with water at a temperature of 22-24°C. The Any-Maze software began to record the state of the mice in the water for 6 minutes. The first 2 minutes were the adaptation stage. The last 4 minutes of immobility of each mouse were analyzed. The mice were considered immobile if they floated in an upright position or raised their heads above the water after only some small movements.

[0086] Antidepressant Model Evaluation V:

[0087] Before the test, the mice were allowed to adapt to the test environment for 30 minutes. The Any-Maze software was used to divide the center area of ​​the open box, set the scale, mouse color and test time, and the camera was turned on for video recording to record the autonomous movement distance, total movement time and center area time of the mice within 5 minutes.

[0088] Antidepressant Model Evaluation VI:

[0089] Enzyme-linked immunosorbent assay (ELISA): Mice were decapitated, and the hippocampus was quickly removed, washed with 4°C saline, dried with filter paper, and quickly frozen in liquid nitrogen for storage; the OD value was determined according to the steps in the kit instructions, and a standard curve was drawn based on the OD value of the standard substance with known concentration, and the 5-HT, NE and GABA contents in the hippocampus tissue of each group of mice were calculated based on the standard curve;

[0090] Antidepressant Model Evaluation VII:

[0091] Golgi staining: Golgi staining was used to observe the synaptic plasticity of the mouse hippocampus. The mouse brain tissue was immersed in the Golgi staining solution for 48 h, and then a new staining solution was replaced, and then a new staining solution was replaced every 3 days for a total of 14 days, and stored at room temperature in the dark. Next, the brain tissue was washed three times in distilled water and then immersed in 80% glacial acetic acid overnight. When the tissue softened, it was washed with distilled water and placed in 30% sucrose. After that, the tissue was cut into 100 μm thick sections and pasted on gelatin slides and dried overnight in the dark. The next day, the dried tissue slides were treated with concentrated ammonia for 15 min, washed with distilled water for 1 min, treated with acidic fixative for 15 min, washed with distilled water for 3 min, and sealed with glycerol gelatin after drying. Photos were taken using a Nikon DS-U3. The analysis of dendrites and dendritic spines in the hippocampus was completed using ImageJ software.

[0092] Example 7. Evaluation of the antidepressant activity of desvenlafaxine-γ-aminobutyric acid

[0093] ICR mice (Laboratory Animal Quality Certificate No.: NO.202370789), male, 22-30g, were randomly divided into control group, model group, desvenlafaxine 16.78mg / kg group (ODV 16.78mg / kg), γ-aminobutyric acid 6.57mg / kg group (GABA6.57mg / kg), desvenlafaxine-γ-aminobutyric acid 34.32mg / kg group (ODV-GABA 34.32mg / kg), n=10 in each group; the control group and the model group were intraperitoneally injected with corresponding doses of normal saline, and the drug treatment group was intraperitoneally administered with corresponding doses of drugs, and the administration volume was 10ml / kg;

[0094] Experimental results:

[0095] (1) Effects of desvenlafaxine-γ-aminobutyric acid on the behavior of mice exposed to chronic unpredictable mild stress

[0096] After 28 days of modeling, the mice were evaluated behaviorally. Figure 4As shown in A, the mean body weights of mice in the control group, model group, desvenlafaxine 16.78 mg / kg group, GABA 6.57 mg / kg group, and desvenlafaxine-GABA 34.32 mg / kg group were 35.09 g, 30.29 g, 33.18 g, 2.74 g, and 34.42 g, respectively; compared with the model group, the body weights of mice in the drug treatment group increased by 8.7%, 8.1%, and 13.63% at 28 days, respectively, and the body weight of mice in the desvenlafaxine-GABA group increased the most; Figure 4 As shown in B, the sucrose preference of mice in the control group, model group, desvenlafaxine 16.78 mg / kg group, GABA 6.57 mg / kg group, and desvenlafaxine-GABA 34.32 mg / kg group at 12 h was 76.70%, 57.21%, 73.13%, 69.72%, and 76.17%, respectively; the sucrose preference value of mice after drug treatment was significantly improved compared with that in the model group; among them, the sucrose preference of mice in the desvenlafaxine-GABA group at 12 h was significantly higher than that in the two single drug groups; as shown in Figure 4 As shown in CD, in the 6-min tail suspension test and forced swimming test of mice, the immobility time of mice in the control group, model group, desvenlafaxine 16.78 mg / kg group, γ-aminobutyric acid 6.57 mg / kg group, and desvenlafaxine-γ-aminobutyric acid 34.32 mg / kg group after 4 minutes was 110.32s / 107.53s, 143.06s / 153.22s, 105.02s / 116.13s, 120.32s / 141.21s, and 74.8s / 110.32s, respectively. The immobility time of mice in the model group was significantly increased compared with that in the control group, indicating that chronic unpredictable mild stress modeling would lead to a decrease in the survival desire of mice and produce behavioral despair. However, after drug treatment, the immobility time of mice decreased, indicating that the survival desire of mice was improved after drug administration.

[0097] The open field test is to explore the curiosity, exploration ability and anxiety of mice; Figure 4 As shown in E, the total distances moved by mice within 5 min were 9.69 m, 5.65 m, 8.56 m, 7.95 m, and 9.06 m in the control group, model group, desvenlafaxine 16.78 mg / kg group, γ-aminobutyric acid 6.57 mg / kg group, and desvenlafaxine-γ-aminobutyric acid 34.32 mg / kg group, respectively. The total distances moved by the drug-treated groups increased by 51.50%, 41.24%, and 60.35%, respectively, compared with the model group. The total distance moved by desvenlafaxine-γ-aminobutyric acid increased the most in the drug-treated groups; Figure 4As shown in Figure F, the movement trajectory of mice in the model group was less than that in the control group, but after treatment in the drug group, the movement activity of mice was significantly improved; Figure 4 As shown in G, the total movement time of mice in the control, model and each drug-treated group within 5 minutes was 122.62s, 72.29s, 118.74s, 117.33s and 123.06s, respectively. It can be seen from the data that when treated with desvenlafaxine-γ-aminobutyric acid, the total movement time of mice was the longest, followed by desvenlafaxine monotherapy and γ-aminobutyric acid monotherapy; Figure 4 As shown in H, the time that mice in the control, model and drug-treated groups stayed in the central area was 31.41s, 15.81s, 20.36s, 21.4s and 24.05s, respectively. The mice in the desvenlafaxine-γ-aminobutyric acid group stayed in the central area the longest, followed by γ-aminobutyric acid and desvenlafaxine, indicating that the exploration desire of mice was enhanced after drug treatment, and the effect of desvenlafaxine-γ-aminobutyric acid was the most significant.

[0098] In summary, in behavioral evaluation, chronic unpredictable mild stress can cause mice to lose weight, decrease sucrose preference, prolong immobility time in tail suspension and forced swimming tests, decrease total moving distance, decrease total moving time, and decrease center zone residence time. However, after treatment with desvenlafaxine, γ-aminobutyric acid or desvenlafaxine-γ-aminobutyric acid sedative, mice gain weight, increase sucrose preference, decrease immobility time in tail suspension test and forced swimming test, increase total moving distance, prolong total moving time, and increase center zone residence time, indicating that the euphoria, despair behavior, curiosity and desire for exploration of mice are improved, and anxiety is alleviated. Among them, desvenlafaxine-γ-aminobutyric acid sedative has obvious advantages over the single drug group in the above behavioral evaluations. Therefore, desvenlafaxine-γ-aminobutyric acid sedative has a good antidepressant and antianxiety effect.

[0099] (2) Effects of desvenlafaxine-γ-aminobutyric acid on neurotransmitters in the brain of mice exposed to chronic unpredictable mild stress

[0100] Studies have shown that the increase in monoamine neurotransmitter levels is related to antidepressant effects. In order to study the antianxiety and antidepressant effects of venlafaxine and γ-aminobutyric acid, Elisa was used to detect the content of monoamine neurotransmitters such as 5-HT, NE, and GABA in the hippocampus. Figure 5 As shown in A, the 5-HT content in the mouse hippocampus of the control, model and each drug-treated group was 46.27 ng / mg, 25.45 ng / mg, 44.64 ng / mg, 40.59 ng / mg and 46.27 ng / mg; Figure 5As shown in B, the contents of NE were 166.17pg / mg, 97.5pg / mg, 146pg / mg, 125.51pg / mg, and 158.06pg / mg; Figure 5 As shown in C, the GABA content was 12.27pg / mg, 5.33pg / mg, 7.55pg / mg, 7.68pg / mg, and 10.38pg / mg; when treated with drugs, the 5-HT, NE, and GABA neurotransmitter contents of mice treated with desvenlafaxine-γ-aminobutyric acid were higher than those of desvenlafaxine alone and γ-aminobutyric acid alone;

[0101] In summary, chronic unpredictable mild stress can cause a decrease in the content of 5-HT, NE and GABA in the hippocampus of mice. After drug treatment, the content of 5-HT, NE and GABA increased. The content of neurotransmitters after desvenlafaxine-γ-aminobutyric acid treatment was better than that of single drug treatment, indicating that desvenlafaxine-γ-aminobutyric acid treatment can significantly reverse the neuronal damage induced by chronic unpredictable mild stress and increase the content of neurotransmitters in the brain.

[0102] (3) Effects of desvenlafaxine-γ-aminobutyric acid on synaptic structural plasticity of neurons in the hippocampal CA1 region of mice subjected to chronic unpredictable mild stress

[0103] To determine whether chronic unpredictable mild stress induces structural damage to synaptic structures of neurons in the hippocampal CA1 region, we performed Golgi staining on mouse brain sections. Figure 6 As shown in AB, the density analysis of single dendritic spines using ImageJ software showed that the density of dendritic spines in the CA1 region of the hippocampus of the control group mice was 9.93 / 10μm, and the density of dendritic spines in the CA1 region of the hippocampal neurons of the model group mice was 5.48 / 10μm. After drug treatment, the dendritic spine density of the CA1 region of the hippocampal neurons of the desvenlafaxine 16.78mg / kg group, the γ-aminobutyric acid 6.57mg / kg group, and the desvenlafaxine-γ-aminobutyric acid group was 7.93 / 10 μm, 5.85 / 10μm, 9.23 / 10μm, the neuronal density of CA1 region in the drug treatment group was increased by 44.7%, 0.67%, and 68.43% respectively compared with the model group. The data showed that the neuronal density of mice treated with desvenlafaxine-γ-aminobutyric acid was most significantly increased, indicating that drug treatment can better restore neuronal damage than monotherapy. In addition, the path of single neuronal branches on the brain slices after Golgi staining was traced, and the Sholl Analysis plug-in was used for analysis and statistics, such as Figure 6CD results showed that compared with the control group, the number of neuronal intersections in the CA1 region of the hippocampus of the model group mice was significantly higher than that of the control group at 0-100μm, among which the number of neuronal intersections was the highest at 50μm, and the number of intersections in the control group, model group and each drug-treated group were 17, 5, 11, 6 and 14, respectively. The results showed that chronic unpredictable mild stress modeling would reduce the complexity of dendrites of neurons in the hippocampus. When treated with desvenlafaxine or γ-aminobutyric acid alone, the complexity of dendrites of neurons in the CA1 region of the hippocampus was improved, but when treated with desvenlafaxine-γ-aminobutyric acid alone, the complexity of dendrites of neurons in the CA1 region of the hippocampus was significantly better than that of the monotherapy group.

[0104] In summary, chronic unpredictable mild stress can cause a decrease in neuronal density and a decrease in the complexity of neuronal dendrites in the CA1 region of the mouse hippocampus. After drug treatment, the neuronal density in the CA1 region of the mouse hippocampus increased and the complexity of neuronal dendrites improved. Among them, the neuronal damage of mice was minimal after treatment with desvenlafaxine-GABA, indicating that desvenlafaxine-GABA is more effective in neuronal repair than monotherapy.

[0105] Example 8. Evaluation of the antidepressant activity of a combination of desvenlafaxine and γ-aminobutyric acid

[0106] ICR mice (Laboratory Animal Quality Certificate No.: NO.202370789), male, 22–30 g, were randomly divided into control group, model group, desvenlafaxine 16.78 mg / kg group (ODV 16.78 mg / kg), γ-aminobutyric acid 6.57 mg / kg group (GABA6.57 mg / kg), desvenlafaxine and γ-aminobutyric acid group (ODV 16.78 mg / kg + GABA6.57 mg / kg), n = 10 in each group; the control group and the model group were intraperitoneally injected with the corresponding dose of normal saline, and the drug treatment group was intraperitoneally administered with the corresponding dose of drug, and the administration volume was 10 ml / kg;

[0107] Experimental Results

[0108] (1) Effects of desvenlafaxine and γ-aminobutyric acid combination on the behavior of mice exposed to chronic unpredictable mild stress

[0109] Mouse behavioral assessment, such as Figure 7As shown in A, the mean weights of mice in the control group, model group, desvenlafaxine 16.78 mg / kg group, γ-aminobutyric acid 6.57 mg / kg group, desvenlafaxine 16.78 mg / kg and γ-aminobutyric acid 6.57 mg / kg groups were 35.09 g, 30.29 g, 33.18 g, 32.74 g, and 33.64 g, respectively; the weights of mice in the drug treatment groups increased by 8.7%, 8.1%, and 9.96% compared with those in the model group, respectively, and the weight of mice increased the most when treated with the combination of desvenlafaxine and γ-aminobutyric acid; Figure 7 As shown in B, the sucrose preference of the control group, model group, desvenlafaxine 16.78 mg / kg group, GABA 6.57 mg / kg group, and desvenlafaxine and GABA combination group were 76.70%, 57.21%, 73.13%, 69.72%, and 74.84%, respectively. Among them, compared with the model group, the sucrose preference value of the combination group increased most significantly, indicating that the euphoria of the mice was improved, followed by the desvenlafaxine and GABA group; Figure 7 As shown in CD, in the tail suspension test and forced swimming test, the immobility time after 4 min in the normal group, model group, desvenlafaxine 16.78 mg / kg group, GABA 6.57 mg / kg group and the combination of desvenlafaxine and GABA were 110.32 s / 107.53 s, 143.06 s / 153.22 s, 105.02 s / 116.13 s, 120.32 s / 141.21 s, 89.91s / 113.68s. Compared with the model group, the immobility time of the drug treatment group in the tail suspension test and forced swimming test decreased by 26.60% / 24.21%, 15.90% / 7.8%, and 37.15% / 25.81%, respectively. The experimental results showed that after drug treatment, the immobility time of the combination of desvenlafaxine and γ-aminobutyric acid decreased most significantly, indicating that the combination treatment better alleviated the despair behavior of mice.

[0110] like Figure 7 As shown in E, in the open field test, the total distances moved by mice in 5 minutes were 9.69m, 5.65m, 8.56m, 7.95m, and 8.74m in the normal group, model group, desvenlafaxine 16.78mg / kg group, γ-aminobutyric acid 6.57mg / kg group, and desvenlafaxine and γ-aminobutyric acid combination group, respectively. Compared with the model group, the total distances moved by mice in the drug treatment group increased by 5.15%, 4.05%, and 5.47%, respectively. The total distance moved by the combination group increased the most, indicating that the activity of mice was enhanced; Figure 7 As shown in F, the movement trajectory of mice, the movement trajectory of mice in the model group was reduced compared with that in the control group, but after treatment with a single drug or a combination of drugs, the movement activity of mice was significantly improved; Figure 7 As shown in G, the total movement time of mice in the control, model and each drug-treated group was 122.62s, 72.29s, 118.74s, 117.33s and 121.68s, respectively. The total movement time of mice in the combination of desvenlafaxine and γ-aminobutyric acid was higher than that in the single-drug desvenlafaxine and γ-aminobutyric acid groups; Figure 7 As shown in H, the time that mice in the control, model and drug-treated groups stayed in the central area was 31.41s, 15.81s, 20.36s, 21.4s and 22.51s, respectively. Among them, mice treated with the combination of desvenlafaxine and γ-aminobutyric acid stayed in the central area the longest, followed by γ-aminobutyric acid alone, indicating that the exploration desire of mice was improved and anxiety was relieved after the combination treatment.

[0111] In summary, in behavioral evaluation, chronic unpredictable mild stress can cause mice to lose weight, decrease sucrose preference, prolong immobility time in tail suspension test and forced swimming test, decrease total moving distance, decrease total moving time and decrease center zone residence time. However, after treatment with desvenlafaxine, γ-aminobutyric acid or a combination of desvenlafaxine and γ-aminobutyric acid, mice gain weight, increase sucrose preference, decrease immobility time in tail suspension test and forced swimming test, increase total moving distance, prolong total moving time and increase center zone residence time. This indicates that the euphoria, despair behavior, curiosity and desire to explore of mice are improved, and anxiety is alleviated. Among them, drug combination treatment has obvious advantages over single drug treatment in the above behavioral evaluations. Therefore, the combination of desvenlafaxine and γ-aminobutyric acid has a good anti-depressant and antianxiety effect.

[0112] (2) Effects of desvenlafaxine and γ-aminobutyric acid combination on neurotransmitters in the brain of mice with chronic unpredictable mild stress

[0113] like Figure 8 As shown in A, the 5-HT content in the mouse hippocampus, the 5-HT content in the control, model and each drug-treated group in the mouse hippocampus was 46.27ng / mg, 25.45ng / mg, 44.64ng / mg, 40.59ng / mg, 47.63ng / mg, the 5-HT content in the drug-treated group was increased by 75.4%, 59.49%, 87.15% respectively compared with the model group; the 5-HT content of the combination of desvenlafaxine and γ-aminobutyric acid increased the most, followed by desvenlafaxine alone and γ-aminobutyric acid alone; as shown in Figure 8As shown in B, the NE content was 166.17pg / mg, 97.5pg / mg, 146pg / mg, 125.51pg / mg, and 158.06pg / mg. The NE content in the drug treatment group was increased by 49.74%, 24.63%, and 62.11% respectively compared with the model group. The NE content of the combination of desvenlafaxine and γ-aminobutyric acid increased the most, followed by desvenlafaxine alone and γ-aminobutyric acid alone; Figure 8 C, the GABA contents of the control, model and each drug-treated group were 12.27pg / mg, 5.33pg / mg, 7.55pg / mg, 7.68pg / mg and 10.38pg / mg, respectively. The GABA contents of the drug-treated group were increased by 41.65%, 44.09% and 94.75% respectively compared with the model group. The GABA content in the combination of desvenlafaxine and γ-aminobutyric acid was higher than that in the single-drug treatment group.

[0114] In summary, CUMS can cause a decrease in the content of 5-HT, NE and GABA in the CA1 region of the mouse hippocampus. After drug treatment, the content of 5-HT, NE and GABA neurotransmitters in the combination is higher than that in the single drug group. The content of desvenlafaxine neurotransmitter is higher than that in the 5-HT and NE indexes than that in the γ-aminobutyric acid group, but in the GABA index, the content of the γ-aminobutyric acid group is slightly higher than that of the desvenlafaxine group. The data show that the combination of desvenlafaxine and γ-aminobutyric acid has a better advantage than the single drug in increasing the content of neurotransmitters in the brain, so the combination has a significant advantage in the treatment of antidepressants.

[0115] (3) Effects of desvenlafaxine and γ-aminobutyric acid combination on synaptic structural plasticity of neurons in the hippocampal CA1 region of mice subjected to chronic unpredictable mild stress

[0116] like Fig. 9 As shown in AB, the density analysis of single dendritic spines by ImageJ software found that the dendritic spine densities in the CA1 region of the hippocampus of the control group, model group and each drug-treated group were 9.93 / 10μm, 5.48 / 10μm, 7.93 / 10μm, 5.85 / 10μm and 8.48 / 10μm, respectively. The neuron density in the drug-treated group was increased by 44.7%, 0.67% and 54.74% compared with the model group, respectively. It can be concluded that the increase in the neuron density of mice was most obvious when the combination was treated. In addition, the Sholl Analysis plug-in was used to track the path of a single neuron branch on the brain slice after Golgi staining, as shown in FIG. Fig. 9 The CD results showed that the number of neuronal intersections was the highest at 50 μm, and the number of intersections of the control group, model group and each drug group was 17, 5, 11, 6 and 13 respectively. It can be seen that the number of intersections of the combination was the highest among the drug groups, so the recovery effect of the combination on neuronal damage was better than that of a single drug.

[0117] In summary, chronic unpredictable mild stress can cause a decrease in neuronal density and a decrease in the complexity of neuronal dendrites in the CA1 region of the mouse hippocampus. After drug treatment, the neuronal density in the CA1 region of the mouse hippocampus increased and the complexity of neuronal dendrites increased. Among them, the neuronal density increased the most and the complexity was higher after treatment with the combination, indicating that the combination of desvenlafaxine and γ-aminobutyric acid has a more significant effect on neuronal repair than single-drug treatment.

[0118] Example 9. Evaluation of the antidepressant activity of the combination of venlafaxine and γ-aminobutyric acid

[0119] ICR mice (Laboratory Animal Quality Certificate No.: NO.202370789), male, 22–30 g, were randomly divided into control group, model group, venlafaxine 20 mg / kg group (VLF 20 mg / kg), γ-aminobutyric acid 7 mg / kg group (GABA 7 mg / kg), venlafaxine and γ-aminobutyric acid combination group (VLF 20 mg / kg+GABA 7 mg / kg), n=11 in each group; the control group and the model group were intraperitoneally injected with corresponding doses of normal saline, and the drug treatment group was intraperitoneally administered with corresponding doses of drugs, and the administration volume was 10 ml / kg.

[0120] Experimental Results

[0121] (1) Effects of venlafaxine and γ-aminobutyric acid combination on the behavior of mice exposed to chronic unpredictable mild stress

[0122] like Fig.10 As shown in A, the mean weights of mice in the control group, model group, venlafaxine 20 mg / kg group, γ-aminobutyric acid 7 mg / kg group, and venlafaxine and γ-aminobutyric acid combination group were 32.15 g, 28.47 g, 31.29 g, 30.79 g, and 31.7 g, respectively; compared with the model group, the weights of the drug treatment groups increased by 9.9%, 8.15%, and 11.34%, respectively, and the weight of mice treated with the combination increased significantly; Fig.10 As shown in B, the sucrose preference of the normal group, model group and drug-treated group was 84.84%, 61.06%, 74.08%, 70.36% and 74.62%, respectively; it can be seen from the data that the sucrose preference of the combination is higher than that of venlafaxine alone and γ-aminobutyric acid alone; Fig.10 As shown in C, in the tail suspension test, the immobility time of mice in the normal group, model group and drug treatment group after 4 minutes was 104.78s, 134s, 106.45s, 120.1s and 98.25s respectively; the immobility time of mice in the combination of venlafaxine and γ-aminobutyric acid was the shortest in drug treatment, indicating that the behavioral despair of mice was better alleviated; Fig.10As shown in D, in the forced swimming test, the immobility time of mice in the normal group, model group and drug treatment group after 4 minutes was 110.69s, 143.96s, 107.66s, 127.17s and 95.89s respectively; the data showed that the immobility time of mice in the forced swimming test was reduced when venlafaxine and γ-aminobutyric acid were given as single drugs or as a combination, indicating that the survival desire of mice was enhanced, and the immobility time of mice after the combination treatment was the lowest, indicating that the combination treatment effect was better than that of single drug treatment;

[0123] like Fig.10 As shown in E, in the open field test, the total distances moved by mice in 5 minutes were 14.16m, 7.24m, 11.07m, 10.62m, and 11.47m for the control group, model group, venlafaxine 20mg / kg group, γ-aminobutyric acid 7mg / kg group, and venlafaxine and γ-aminobutyric acid combination group, respectively. The total distances moved by mice during treatment with each drug increased by 52.90%, 46.69%, and 58.43% respectively compared with the model group; the total distance moved by the venlafaxine and γ-aminobutyric acid combination group was the highest, indicating that the motor activity of mice was the best during treatment with the combination group; Fig.10 As shown in F, the movement trajectory of mice, the movement trajectory of mice in the model group was reduced compared with that in the control group, but after treatment in the drug group, the movement activity of mice was significantly improved; Fig.10 As shown in G, the total movement time of mice in the control, model and each drug group was 166.21s, 96.14s, 133.68s, 136.69s and 142.89s, respectively. The total movement time of mice in the combination of venlafaxine and γ-aminobutyric acid was significantly higher than that in the single drug group; Fig.10 As shown in H, the time that mice in the control, model and each drug-treated group stayed in the central area was 23.14s, 15.56s, 24.28s, 28.04s and 24.9s, respectively. The mice in the treatment group with the combination of venlafaxine and γ-aminobutyric acid stayed in the central area the longest, indicating that the mice in the treatment group with the combination had the strongest desire to explore.

[0124] In summary, in behavioral evaluation, chronic unpredictable mild stress can cause mice to lose weight, decrease sucrose preference, prolong immobility time in tail suspension test and forced swimming test, decrease total moving distance, decrease total moving time, and decrease center zone residence time. However, after treatment with venlafaxine, γ-aminobutyric acid, or a combination of venlafaxine and γ-aminobutyric acid, mice gain weight, increase sucrose preference, decrease immobility time in tail suspension and forced swimming test, increase total moving distance, prolong total moving time, and increase center zone residence time. The above behavioral studies show that the euphoria, despair behavior, curiosity and desire for exploration of mice are improved, and anxiety is alleviated. Among them, the combination of venlafaxine and γ-aminobutyric acid has obvious advantages over single drugs in behavioral evaluation. Therefore, the combination of venlafaxine and γ-aminobutyric acid has a good antidepressant and antianxiety effect.

[0125] (2) Effects of venlafaxine and γ-aminobutyric acid combination on neurotransmitters in the brain of mice with chronic unpredictable mild stress

[0126] like Fig.11 As shown in A, the 5-HT content in the hippocampus of mice in the control group, model group, venlafaxine 20 mg / kg group, γ-aminobutyric acid 7 mg / kg group, and venlafaxine and γ-aminobutyric acid combination was 37.38 ng / mg, 21.48 ng / mg, 30.09 ng / mg, 28.14 ng / mg, and 33.90 ng / mg, and the 5-HT content of mice in each drug treatment group was increased by 40.08%, 31%, and 57.82% respectively compared with the model group, and the 5-HT content of venlafaxine and γ-aminobutyric acid combination was higher than that of venlafaxine group and γ-aminobutyric acid group; Fig.11 The NE contents shown in B were 118.24pg / mg, 82.1pg / mg, 109.03pg / mg, 98.35pg / mg, and 110.45pg / mg, and the NE contents of each drug treatment group were increased by 32.8%, 19.79%, and 34.53% respectively compared with the model group. The NE content of the combination of venlafaxine and γ-aminobutyric acid was the highest, followed by the venlafaxine group and the γ-aminobutyric acid group; Fig.11 C, GABA contents in each group were 12.72pg / mg, 7.34pg / mg, 11.48pg / mg, 10.71pg / mg, and 11.71pg / mg, respectively. GABA contents in each drug treatment group increased by 56.4%, 45.91%, and 59.54% respectively compared with the model group. The GABA content in the combination of venlafaxine and γ-aminobutyric acid was the highest, followed by the venlafaxine group and the γ-aminobutyric acid group.

[0127] In summary, chronic unpredictable mild stress can cause a decrease in the levels of 5-HT, NE and GABA in the hippocampus of mice. When treated with drugs, the neurotransmitter content of the combination was higher than that of the single-drug group in the 5-HT, NE and GABA indicators, and venlafaxine was higher than γ-aminobutyric acid. The results showed that both single-drug and combination treatments can increase the neurotransmitter content in the brain, but the combination treatment effect is better.

[0128] (3) Effects of venlafaxine and γ-aminobutyric acid combination on synaptic structural plasticity of neurons in the hippocampal CA1 region of mice subjected to chronic unpredictable mild stress

[0129] like Fig.12 As shown in AB, the density analysis of single dendritic spines by ImageJ software found that the dendritic spine densities in the CA1 region of the mouse hippocampus of the control, model and drug-treated groups were 7.13 / 10μm, 3.39 / 10μm, 5.8 / 10μm, 4.44 / 10μm and 6.15 / 10μm, respectively. The neuronal density of the drug-treated group was increased by 41.56%, 30.97% and 81.42% compared with the model group, respectively. In addition, the Sholl Analysis plug-in was used to track the path of single neuronal branches on the Golgi-stained brain slices, as shown in Figure 1. Fig.12 The CD results showed that the number of neuronal intersections in the control group was smaller than that in the model group within 0-100 μm. However, after drug treatment, the number of neuronal intersections in the CA1 region of the mouse hippocampus increased, and the number of intersections in the combination treatment group was higher than that in the single drug treatment group, which also indicated that the complexity of neuronal dendrites in the combination group was enhanced.

[0130] In summary, CUMS can cause a decrease in neuronal density and a decrease in the complexity of neuronal dendrites in the CA1 region of the mouse hippocampus. After drug treatment, the neuronal density in the CA1 region of the mouse hippocampus increases and the complexity of neuronal dendrites improves. Among them, after treatment with the combination of venlafaxine and γ-aminobutyric acid, the neuronal density in the CA1 region of the mouse hippocampus increases the most, and the neuronal dendrite complexity is higher, indicating that the combination treatment has a better effect on neuronal repair than the single-drug treatment.

Claims

1. A type of synergistic antidepressant drug, characterized in that: The spasticity drug is a compound formed by condensation connection of desvenlafaxine and γ-aminobutyric acid or a pharmaceutically acceptable salt or a pharmaceutically acceptable carrier thereof, and its chemical structure is shown in Formula I:

2. The method for preparing a synergistic antidepressant drug according to claim 1, characterized in that: The preparation steps are as follows: Step (1): protecting the amino group in the γ-aminobutyric acid structure with di-tert-butyl dicarbonate or benzyl chloroformate to obtain intermediate I; Step (2): subjecting desvenlafaxine to a condensation reaction with the intermediate I obtained in the reaction in step (1) under EDCI / DMAP to obtain an intermediate II; Step (3): removing the protecting group of the intermediate II obtained in step (2) under acidic or palladium-carbon conditions to finally obtain the convulsion drug; The specific synthetic route is as follows:

3. A composition of a spasticity drug, comprising the compound formed by condensation connection of desvenlafaxine and γ-aminobutyric acid as claimed in claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

4. A composition, characterized in that The composition comprises desvenlafaxine and γ-aminobutyric acid or venlafaxine and γ-aminobutyric acid.

5. A composition according to claim 4, characterized in that The weight ratio of desvenlafaxine to gamma-aminobutyric acid is 0.1-5:1; the weight ratio of venlafaxine to gamma-aminobutyric acid is 0.1-5:

1.

6. Use of the compound formed by condensation connection of desvenlafaxine and γ-aminobutyric acid as claimed in claim 1 or a pharmaceutically acceptable salt thereof, the combination of the anticonvulsant as claimed in claim 3 or the combination as claimed in claim 4 in the preparation of a drug for preventing or treating depression.

7. The use according to claim 6, characterized in that: The dosage of desvenlafaxine or a pharmaceutically acceptable salt thereof is 10 to 375 mg of desvenlafaxine, the dosage of γ-aminobutyric acid or a pharmaceutically acceptable salt thereof is 2 to 100 mg of γ-aminobutyric acid, and the dosage of venlafaxine or a pharmaceutically acceptable salt thereof is 10 to 225 mg of venlafaxine.

8. The use according to claim 7, characterized in that: The depression described is major depression, bipolar depression, psychotic depression, reactive depression, menopausal depression, childhood depression, secondary depression, postpartum depression, seasonal depression, persistent depression, depression in postmenopausal and perimenopausal women, anxiety depression and panic disorder.

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