Preparation and use of a class of synergistic anti-depression drugs and compositions thereof

Abstract

The application discloses a kind of having synergistic antidepressant effect of twin drug and its composition preparation, application;Belong to medical field, the twin drug is norvenlafaxine-gamma-aminobutyric acid, such as formula I;The pharmaceutical composition includes norvenlafaxine and gamma-aminobutyric acid pharmaceutical composition, venlafaxine and gamma-aminobutyric acid pharmaceutical composition;It also includes using norvenlafaxine-gamma-aminobutyric acid twin drug, norvenlafaxine and gamma-aminobutyric acid pharmaceutical composition and venlafaxine and gamma-aminobutyric acid pharmaceutical composition treatment depression;The purpose of twin drug or its pharmaceutically acceptable salt, composition or twin drug composition in preparation prevention or treatment of depression;Norvenlafaxine-gamma-aminobutyric acid twin drug, norvenlafaxine and gamma-aminobutyric acid pharmaceutical composition and venlafaxine and gamma-aminobutyric acid pharmaceutical composition prevent or treat depression, especially for depression with anxiety, twin drug or composition can improve drug efficacy, and have better anti-anxiety, antidepressant effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and relates to a kind of drug with synergistic antidepressant effect and preparation and application of its composition. BACKGROUND

[0002] Depression is a common mental disorder, involving long-term low mood or loss of pleasure or interest in activities. Depression is one of the highest incidence of mental illness in the world, according to the World Health Organization, there are more than 300 million people with depression in the world, and nearly 800,000 people die from depression every year. Depression accounts for 10% of the total burden of non-fatal diseases in the world, and is expected to become the first global disease burden by 2030.

[0003] Venlafaxine (VLF) is a phenethylamine derivative, a second-generation chiral antidepressant drug. Studies have shown that small-dose treatment of venlafaxine mainly inhibits 5-HT reuptake, and large-dose treatment inhibits 5-HT and NE reuptake, and its structure is shown as formula II. Its main active metabolite is O-desmethylvenlafaxine (ODV), and its structure is shown as 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

[0005] The purpose of the present application is to provide a kind of drug with synergistic antidepressant effect and preparation and application of its composition.

[0006] Technical solution: the drug according to the present application is a compound or its pharmaceutically acceptable salt formed by condensation of desmethylvenlafaxine and gamma-aminobutyric acid, or a pharmaceutically acceptable carrier, and its chemical structure is shown as formula I.

[0007]

[0008] A composition containing desmethylvenlafaxine and gamma-aminobutyric acid, or venlafaxine and gamma-aminobutyric acid.

[0009] A composition of a drug containing the compound or its pharmaceutically acceptable salt and a pharmaceutically acceptable carrier.

[0010] A preparation method of a kind of drug with synergistic antidepressant effect: the method comprises the following steps:

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

[0012] Step 2: condensing nortriptyline with intermediate I obtained in the first step under EDCI / DMAP to obtain intermediate II;

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

[0014]

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

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

[0017] Further, the dosage of nortriptyline or its pharmaceutically acceptable salt is 10-375 mg of nortriptyline, the dosage of γ-aminobutyric acid or its pharmaceutically acceptable salt is 2-100 mg of γ-aminobutyric acid base, and the dosage of nortriptyline or its pharmaceutically acceptable salt is 10-225 mg of nortriptyline base.

[0018] Further, the application of a nortriptyline-γ-aminobutyric acid conjugate or its composition, a nortriptyline-γ-aminobutyric acid composition, a nortriptyline-γ-aminobutyric acid composition, the use of the compound or its pharmaceutically acceptable salt, the composition or the composition of the conjugate in the preparation of a drug for preventing or treating depression.

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

[0020] According to in vivo microdialysis studies, venlafaxine and O-desmethylvenlafaxine rapidly penetrate the brain, increasing hypothalamic norepinephrine concentrations, but not dopamine concentrations, and thus have a highly selective effect on serotonin and norepinephrine monoamine transporters. Therefore, the mechanism of action of venlafaxine and O-desmethylvenlafaxine is to inhibit the serotonin and norepinephrine transporters at 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 at the postsynaptic membrane, and activating the postsynaptic receptors involved in the regulation of emotional state. Venlafaxine and O-desmethylvenlafaxine are now used to treat major depressive disorder, persistent depressive disorder, pediatric depression, postmenopausal and perimenopausal depression, and other diseases, but the drug has the disadvantages of 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 present in microorganisms, plants and vertebrates. γ-Aminobutyric acid is the main inhibitory neurotransmitter in the brain, which maintains homeostasis by balancing the excessive excitability of neurons associated with diseases such as seizures and anxiety. After the activation of γ-aminobutyric acid A receptors, it selectively allows chloride ions to pass through, increases the opening frequency of chloride ion channels, causes hyperpolarization of neurons, and reduces the excitability of the membrane of nerves or other tissues, thereby inhibiting signal transmission. GABAergic transmission occurs in interneurons, which can regulate local neurotransmission, including norepinephrine, dopamine and 5-hydroxytryptamine neurons, so its physiological role is related to regulating synaptic transmission, promoting neuronal development and relaxation, preventing insomnia, 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 γ-aminobutyric acid in the brain of patients with depression is reduced, and animal experiments have also shown that enhancing the activity of γ-aminobutyric acid neurons and increasing the level of γ-aminobutyric acid neurotransmitters has an antidepressant effect in depression models.

[0022]

[0023] Twin drug refers to a new multifunctional molecule formed by covalently combining two same or different lead compounds or drugs, which produces synergistic effect, enhances activity or produces new pharmacological activity, or improves the selectivity of action in vivo. The two molecules combined into a twin drug can have the same type of pharmacological effect, such as aspirin (Aspirin) and acetaminophen (Paracetamol) both have antipyretic and analgesic activity. The new molecule benorilate obtained by esterification of the two has synergistic effect, which not only solves the acid stimulation of aspirin to the stomach, but also enhances the drug efficacy. In addition, two drugs with different pharmacological effects can also be combined to form a twin drug to produce new or combined effects. For example, chlorambucil (Chlorambucil) is an antitumor drug, but it has high toxicity. By designing a steroid as a carrier, the targeting property can be increased. With this idea, prednisolone (Prednisolone) and chlorambucil are combined to form an antitumor drug prednimustine (Prednimustine), which reduces the toxicity of chlorambucil.

[0024] The beneficial effects of the present application are that the norepinephrine reuptake inhibitor, the norepinephrine reuptake inhibitor and GABA combination, the venlafaxine and GABA combination of the present application have synergistic antidepressant and anxiolytic effects in the treatment of depression. The effective dose of a single component can be effectively reduced by different antidepressant mechanisms; the synergistic antidepressant and anxiolytic effects between the above-mentioned drugs are confirmed by the equimolar method of the present application, which has no obvious toxic and side effects while exerting antidepressant and anxiolytic effects, and has good clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

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

[0026] Figure 2 is the hydrogen spectrum of 4-(2-(dimethylamino)-1-(1-hydroxycyclohexyl)ethyl)phenyl 4-((tert-butoxycarbonyl)amino)butanoate (intermediate II) in the embodiment of the present application;

[0027] Figure 3 is the hydrogen spectrum of 4-(2-(dimethylamino)-1-(1-hydroxycyclohexyl)ethyl)phenyl 4-aminobutanoate (norepinephrine reuptake inhibitor-GABA) in the embodiment of the present application;

[0028] Figure 4 Figure 1 is a schematic diagram of the effect of nortriptyline-gamma-aminobutyric acid combination on the behavior of mice in a chronic unpredictable mild stress model according to an embodiment of the present application. Figure 4 A: 28d body weight change; Figure 4 B: 12h sucrose preference test; Figure 4 C: tail suspension test for 4 min; Figure 4 D: forced swimming test for 4 min; Figure 4 E: total movement distance of mice in 5 min in the open field test; Figure 4 F: movement trajectory of mice in 5 min in the open field test; Figure 4 G: total movement time of mice in 5 min in the open field test; Figure 4 H: time of mice in the center area in 5 min in the open field test;

[0029] Figure 5 Figure 2 is a schematic diagram of the effect of nortriptyline-gamma-aminobutyric acid combination on neurotransmitters in the brain of mice in a chronic unpredictable mild stress model according to an embodiment of the present application. Figure 5 A: serotonin content in the hippocampus of mice; Figure 5 B: norepinephrine content in the hippocampus of mice; Figure 5 C: gamma-aminobutyric acid content in the hippocampus of mice;

[0030] Figure 6 Figure 3 is a schematic diagram of the effect of nortriptyline-gamma-aminobutyric acid combination on synaptic structural plasticity of neurons in the CA1 region of the hippocampus of mice in a chronic unpredictable mild stress model according to an embodiment of the present application. Figure 6 A: representative image of dendritic spine density in the CA1 region of the hippocampus of mice; Figure 6 B: analysis of dendritic spine density in the CA1 region of the hippocampus of mice; Figure 6 C: neuron branch path tracing graph in the CA1 region of the hippocampus of mice; Figure 6 D: analysis of neuron dendritic complexity in the CA1 region of the hippocampus of mice;

[0031] Figure 7 Figure 4 is a schematic diagram of the effect of nortriptyline-gamma-aminobutyric acid combination on the behavior of mice in a chronic unpredictable mild stress model according to an embodiment of the present application. Figure 7 A: 28d body weight change; Figure 7 B: 12h sucrose preference test; Figure 7 C: tail suspension test for 4 min; Figure 7 D: forced swimming test for 4 min; Figure 7 E: total movement distance of mice in 5 min in the open field test; Figure 7 F: movement trajectory of mice in 5 min in the open field test; Figure 7 G: total movement time of mice in 5 min in the open field test; Figure 7H: Time in the center zone of the open field test for 5 min;

[0032] Figure 8 Figure 2 is a schematic diagram of the effect of the O-desmethylvenlafaxine and GABA composition of the present application on neurotransmitters in the brain of a mouse model of chronic unpredictable mild stress; Figure 8 A: 5-hydroxytryptamine content in the hippocampus of a mouse; Figure 8 B: norepinephrine content in the hippocampus of a mouse; Figure 8 C: GABA content in the hippocampus of a mouse);

[0033] Figure 9 Figure 3 is a schematic diagram of the effect of the O-desmethylvenlafaxine and GABA composition of the present application on synaptic structural plasticity of neurons in the CA1 region of the hippocampus of a mouse model of chronic unpredictable mild stress; Figure 9 A: representative image of dendritic spine density in the CA1 region of the hippocampus of a mouse; Figure 9 B: analysis of dendritic spine density in the CA1 region of the hippocampus of a mouse; Figure 9 C: neuron branch path tracing graph in the CA1 region of the hippocampus of a mouse; Figure 9 D: neuron dendritic complexity analysis in the CA1 region of the hippocampus of a mouse);

[0034] Figure 10 Figure 4 is a schematic diagram of the effect of the venlafaxine and GABA composition of the present application on behavior of a mouse model of chronic unpredictable mild stress; Figure 10 A: 28d body weight change; Figure 10 B: 12h sucrose preference test; Figure 10 C: tail suspension test for the last 4 min; Figure 10 D: forced swimming test for the last 4 min; Figure 10 E: total movement distance of a mouse in the open field test for 5 min; Figure 10 F: movement trajectory of a mouse in the open field test for 5 min; Figure 10 G: total movement time of a mouse in the open field test for 5 min; Figure 10 H: time in the center zone of the open field test for 5 min;

[0035] Figure 11 Figure 5 is a schematic diagram of the effect of the venlafaxine and GABA composition of the present application on neurotransmitters in the brain of a mouse model of chronic unpredictable mild stress; Figure 11 A: 5-hydroxytryptamine content in the hippocampus of a mouse; Figure 11 B: norepinephrine content in the hippocampus of a mouse; Figure 11 C: GABA content in the hippocampus of a mouse);

[0036] Figure 12Figure 1 is a schematic diagram showing the effect of the inventive venlafaxine and gamma-aminobutyric acid combination on synaptic structural plasticity of hippocampal CA1 neurons of mice in a chronic unpredictable mild stress model Figure 12 A: Representative images of dendritic spine density in the hippocampal CA1 region of mice; Figure 12 B: Analysis of dendritic spine density in the hippocampal CA1 region of mice; Figure 12 C: Dendritic arbor tracing of neurons in the hippocampal CA1 region of mice; Figure 12 D: Analysis of dendritic complexity of neurons in the hippocampal CA1 region of mice. DETAILED DESCRIPTION

[0037] In some embodiments, the inventive combination can be used in combination with at least one other therapeutic agent selected from the group consisting of anti-schizophrenic agents, anti-anxiety agents, anti-depressants, anti-epileptic agents, anti-Alzheimer's disease agents. The other therapeutic agent can be administered prior to, concurrently with, or after administration of the inventive combination.

[0038] The inventive norelafaxine-gamma-aminobutyric acid combination and pharmaceutically acceptable excipients are formulated into a form suitable for administration by any appropriate route, and the active combination is preferably in unit dosage form, or in a form that the patient can self-administer in a single dose. The inventive combination can be presented in unit dosage form as a tablet, capsule, injection, granule, tincture, lozenge, suppository, reconstituted powder or liquid preparation.

[0039] The dosage of the inventive combination used in the method of use will generally vary with the severity of the disease, the body weight of the patient, and the relative potency of the compound. As a general guide, a suitable unit dosage can be 0.01-1000 mg.

[0040] The present application also provides a method for treating depression, which comprises administering to a subject in need thereof a therapeutically effective amount of the inventive compound. In the present application, the treatment of depression includes, but is not limited to, anti-depression related symptoms, amelioration of depression related symptoms, treatment of depression related symptoms, prevention of depression related symptoms. In some embodiments, the administration of the inventive combination or pharmaceutical composition can improve the therapeutic activity compared to the administration of a single moiety alone. In certain embodiments, the inventive combination or pharmaceutical composition is administered in combination with other drugs. The other drugs can be administered prior to, concurrently with, or after administration of the inventive compound or pharmaceutical composition.

[0041] EXAMPLE

[0042] Example 1, Synthesis of Norelafaxine-gamma-aminobutyric acid (ODV-GABA)

[0043] Reaction Scheme:

[0044]

[0045] Intermediate I was obtained by reaction of gamma-aminobutyric acid with di-tert-butyl dicarbonate, and further reacted with nortriptyline to form intermediate II, and finally the target product nortriptyline-gamma-aminobutyric acid was obtained under acidic conditions;

[0046] Firstly, in a 100 mL round-bottom flask, 5.0 g (48.48 mmol, 1.0 eq) of gamma-aminobutyric acid, 40 mL of water, 3.87 g (96.75 mmol, 2.0 eq) of sodium hydroxide were added under stirring, and 12.69 g (58.14 mmol, 1.2 eq) of di-tert-butyl dicarbonate was added dropwise. After the dropwise addition was completed, the reaction was carried out at room temperature for 4 h, and TLC monitoring showed that the reaction was complete. After extraction with 15 mL x 3 dichloromethane, the pH was adjusted to about 3 to 4 with 1 mol / L hydrochloric acid solution, and then extracted with 15 mL x 3 dichloromethane. The organic phase was dried and concentrated to obtain 8.67 g of intermediate I as a white oil with a yield of 88.29%. 1 H NMR (500 MHz, Chloroform-d) δ 4.78 (s, 1H), 3.77-3.70 (m, 1H), 3.19 (t, J = 7.0 Hz, 2H), 2.43-2.34 (m, 2H), 1.83 (m, J = 7.0 Hz, 2H), 1.45 (s, 9H). (-)-HR-ESI-MS m / z 203.11576 (calcd. 202.11522 for C9H 16 NO4 - [M-H] - );

[0047] Secondly, in a 100 mL round-bottom flask, 5 g (24.6 mmol, 1.3 eq) of intermediate I, 5 g (18.98 mmol, 1.0 eq) of nortriptyline, 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, and the reaction was carried out at room temperature for 3 h. TLC monitoring showed that the reaction was basically complete. After extraction with 50 mL of water, the organic phase was dried and concentrated. Column chromatography (dichloromethane:methanol = 50:1) of the organic phase gave 6.21 g of intermediate II as a white solid with a yield of 64.59%. 1H NMR (500 MHz, Chloroform-d) δ 7.12 (d, J = 8.6 Hz, 2H), 6.99 (d, J = 8.6 Hz, 2H), 4.67 (s, 1H), 3.27 (q, J = 14.6, 13.5 Hz, 3H), 2.99 (m, J = 12.4, 3.3 Hz, 1H), 2.60 (t, J = 7.3 Hz, 2H), 2.31 (s, 6H), 2.29 (d, J = 3.3 Hz, 1H), 1.92 (m, J = 7.0 Hz, 2H), 1.76 - 1.60 (m, 3H), 1.53 (q, J = 13.9 Hz, 3H), 1.44 (s, 9H), 1.40 - 1.22 (m, 3H), 0.98 - 0.81 (m, 2H). (+)-HR-ESI-MS m / z 448.29372 (calcd. 449.29451 for C 25 H 41 N2O5 + [M+H] + )

[0048] Finally, 0.35 g of intermediate II was put into a 25 mL round bottom flask, 5 mL of 2M HC1 / EA solution was added dropwise, and the reaction was carried out at room temperature under nitrogen protection; TLC monitoring after 2 h showed that the reaction was complete; 5 mL of water was added, and the pH was adjusted to about 9-10 with saturated sodium bicarbonate aqueous solution, and 8 mL of dichloromethane was extracted for 3 times, and the organic phase was dried and concentrated, and then column chromatography (dichloromethane:methanol = 45:1) was used to obtain O-desmethylvenlafaxine-gamma-aminobutyric acid white solid 0.2 g, yield: 73%. 1 H NMR (500 MHz, DMSO-d6) δ 7.40 (d, J = 8.1 Hz, 2H), 7.12 (d, J = 8.0 Hz, 2H), 3.66 (s, 1H), 3.53 (s, 1H), 3.16 (s, 2H), 2.86 (q, J = 6.7 Hz, 2H), 2.73 (t, J = 7.4 Hz, 2H), 2.68 (d, J = 4.6 Hz, 3H), 2.61 (d, J = 4.5 Hz, 3H), 1.93 (t, J = 7.6 Hz, 2H), 1.65 (d, J = 13.2 Hz, 1H), 1.56 (d, J = 11.4 Hz, 1H), 1.43 (d, J = 8.9 Hz, 3H), 1.34 (s, 1H), 1.27 - 1.13 (m, 4H), 1.10 - 0.92 (m, 2H). (+)-HR ESI-MS: m / z 349.2492 (calcd. 349.2486 for C 20 H 33 N2O3 + [M+H] + )

[0049] Example 2, Synthesis of O-desmethylvenlafaxine-γ-aminobutyric acid (ODV-GABA)

[0050] In 50 mL round bottom flask, charged with 1 g of intermediate II, dichloromethane 5 mL, trifluoroacetic acid 3 mL was added drop wise at 0 °C, TLC monitored the reaction completion after 1 h, column chromatography (dichloromethane: methanol = 40: 1) gave O-desmethylvenlafaxine-γ-aminobutyric acid trifluoroacetate salt, white solid 0.58 g, yield: 56.31 %.

[0051] Example 3, Synthesis of O-desmethylvenlafaxine-γ-aminobutyric acid (ODV-GABA)

[0052] In 50 mL round bottom flask, charged with 1 g of intermediate II, dichloromethane 5 mL, trifluoroacetic acid 3 mL was added drop wise at 0 °C, TLC monitored the reaction completion after 1 h, column chromatography (dichloromethane: methanol = 40: 1) gave O-desmethylvenlafaxine-γ-aminobutyric acid trifluoroacetate salt, white solid 0.58 g, yield: 56.31 %.

[0053] Example 4, Synthesis of O-desmethylvenlafaxine-γ-aminobutyric acid (ODV-GABA)

[0054] In 50 mL round bottom flask, charged with 1 g (2.22 mmol, 1 eq) of intermediate II, ethyl acetate 10 mL, oxalic acid 0.29 g (2.3 mmol, 1.05 eq) in ethyl acetate 10 mL was added drop wise; after 2 h, the filter cake was washed with ethyl acetate, dried under vacuum at 50 °C for 5 h to give O-desmethylvenlafaxine-γ-aminobutyric acid oxalate salt, white solid 1.2 g, yield 81.67 %.

[0055] Example 5, Synthesis of O-desmethylvenlafaxine-γ-aminobutyric acid (ODV-GABA)

[0056] Reaction scheme:

[0057]

[0058] First, in a 250 mL round-bottom flask, γ-aminobutyric acid 5 g (48.48 mmol, 1 eq), water 50 mL, slowly add sodium carbonate 5.13 g (48.4 mmol, 1 eq) at 0 °C, stirring; slowly drop 30 mL of a solution of CbzCl 9.92 g (58.15 mmol, 1.2 eq) in 1,4-dioxane; after the drop is complete, stir the reaction at room temperature for 3 h, TLC monitoring shows that the reaction is substantially complete; extract with ethyl acetate 50 mL x 3, adjust the pH of the aqueous phase to about 2-3 with 1 mol / L hydrochloric acid solution, extract with ethyl acetate 50 mL x 3, dry and concentrate the organic phase to obtain intermediate III as a white oil 6.55 g, yield 57%.

[0059] Then, in a 100 mL round-bottom flask, add 5 g (24.6 mmol, 1.3 eq) of intermediate III, desvenlafaxine 5 g (18.98 mmol, 1.0 eq), DMAP 0.23 g (18.82 mmol, 0.1 eq), EDCI 4.36 g (22.74 mmol, 1.2 eq), dichloromethane 50 mL, react at room temperature for 3 h, TLC monitoring shows that the reaction is substantially complete; add 50 mL of water, extract, dry and concentrate the organic phase, column chromatography (dichloromethane:methanol = 50:1) to obtain intermediate IV as a white solid 6.21 g, yield 64.42%.

[0060] Finally, in a 50 mL round-bottom flask, add 1 g (2.07 mmol, 1 eq) of intermediate IV, Pd / C 10% 0.02 g, ammonium formate 0.39 g (6.18 mmol, 3 eq), methanol 15 mL, reflux at 80 °C for 4 h; filter the reaction liquid, add 10 mL of water to the filtrate, extract with dichloromethane 20 mL x 2, dry and concentrate the organic phase, column chromatography (dichloromethane:methanol = 30:1) to obtain desvenlafaxine-γ-aminobutyric acid 0.13 g, yield 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 Dongfang breeding farm (experimental animal quality certificate number: NO.202370789); all animal facilities and feeding methods meet national standards; the laboratory temperature is 21±3 °C, the humidity is 50±20%, and the animals are fed under 12 h light-dark alternation; the animals are fasted for 12 h before blood sampling but can drink water; the chromatographic column is AQ-C18, (2.1 x 50 mm, 5 μm); the mobile phase is A: 0.1% formic acid-water; B: 0.1% formic acid-acetonitrile, the injection volume is 10 μl

[0063] The experimental process is as follows:

[0064] (1) Take the appropriate amount of blank plasma in the EP tube, then add 100 μM working solution, the final concentration is 1 μM, vortex mix;

[0065] (2) The sample was placed in a 37 °C water bath, parallel 2 copies; Incubation time points are 0, 5, 15, 30, 60 min;

[0066] (3) Compound: at the time point from the incubation system to take 20 μl sample, add 300 μl internal standard precipitant, vortex mix, then the sample centrifuged 10 min (5500g), then take 150 μl supernatant plus 150 μl ultrapure water in 96 well plate;

[0067] (4) Control: at the time point from the incubation system to take 20 μl sample, add 300 μl internal standard precipitant, vortex mix, then the sample centrifuged 10 min (5500g), then take 150 μl supernatant plus 150 μl ultrapure water in 96 well plate;

[0068] (5) Absolute 0 point (compound): take 18 μl sample blank plasma, add 300 μl internal standard precipitant, add 10 μM test substance working solution 2 μl, vortex mix, then the sample centrifuged 10 min (5500g), then take 150 μl supernatant plus 150 μl ultrapure water in 96 well plate;

[0069] (6) vortex mix, LC-MS / MS sample analysis.

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

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

[0072]

[0073] Pharmacological examples

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

[0075] Chronic unpredictable mild stress model is to simulate the pathogenesis of human depression environment, research on the pathogenesis of depression, and provide experimental basis for the screening and efficacy evaluation of antidepressants; The model is considered to be able to effectively induce depression-like symptoms, and the behavior characteristics of the animals after stress will change, and the model is persistent and stable, which has high value as a depression model, and has great significance for the screening of depression drugs and the research of depression mechanism.

[0076] In this experiment, the experimental method of modeling and drug administration at the same time was used, and the mice were modeled and administered for 28 days. Except for the control group, each group of mice was modeled after administration every day. The normal control group of mice was placed in a room with suitable temperature, humidity and light conditions, and no stimulation was given except for 12h fasting and water deprivation before sucrose preference. The remaining modeling group mice received continuous stress stimulation for 4 weeks, a total of 11 stress sources, including tail clamping (2-5min), cold water swimming (12℃, 6-10min), noisy sound (5-12h), crowding (2-5h), restraint (0.5-2h), cage tilt (45°, 5-12h), no bedding (5-12h), wet bedding (2-12h), night light (12h), fasting (6-12h), and water deprivation (6-12h). In order to maximize the unpredictability of the program, the duration and intensity of different stressors were different. The stressors started at 9:00 to 21:00 in the day, and 2-3 stressors were randomly selected each day with at least 2h rest time between two stressors. The analysis of chronic unpredictable mild stress model was mainly carried out by behavioral evaluation. Common evaluation methods include sucrose preference test, tail suspension test, forced swimming test, open field test, etc. In addition, it can also be evaluated by neurotransmitter content and neuron damage.

[0077] Antidepressant model evaluation

[0078] Antidepressant model evaluation one:

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

[0080] Antidepressant model evaluation two:

[0081] Sugar water preference test (SPT): After 4 weeks of continuous modeling and drug administration, the sugar water preference of mice was tested. All mice were uniformly housed in a single cage. Each mouse was given a 1% sucrose solution and a pure water bottle for sugar water training for 48h before testing. Then the mice were fasted and water deprived for 24h, and finally tested for 12h. The positions of the sugar water bottle and the pure water bottle were exchanged in the middle of the test, and the sugar consumption and water consumption in 12h were recorded. Sugar water preference index = sucrose consumption / (sucrose consumption + water consumption) * 100%;

[0082] Antidepressant model evaluation three:

[0083] Tail suspension test (TST): test after 4 weeks of continuous administration; first open the behavior program, select the background with obvious contrast with the color of the mouse, white mouse with black background; second, the tail of the mouse is fixed with tape, suspended on the support, the head is about 30 cm away from the table; Any-Maze software starts recording the state of the mouse in the suspension for 6 min, the first 2 min is the adaptation stage, and the last 4 min of each mouse is analyzed; keep the environment quiet during the experiment to avoid external interference;

[0084] Antidepressant model evaluation four:

[0085] Forced swimming test (FST): test after 4 weeks of continuous administration; put the mouse into a transparent glass container with water, 28 cm high and 15 cm in diameter, water temperature 22-24℃, Any-Maze software starts recording the state of the mouse in the water for 6 min, the first 2 min is the adaptation stage, and the last 4 min of each mouse is analyzed; the mouse is considered to be immobile if it stands upright or only makes small movements to expose its head above the water surface;

[0086] Antidepressant model evaluation five:

[0087] Before the test, the mouse is adapted to the test environment for 30 min, and Any-Maze software is used to divide the center area of the open box, set the scale, mouse color and test time, the camera starts the video recording function, and records the self-moving distance of the mouse within 5 min, total moving time, center area time;

[0088] Antidepressant model evaluation six:

[0089] Enzyme-linked immunosorbent assay (ELISA): the mouse is decapitated and killed, the hippocampus is quickly stripped, washed with physiological saline at 4℃ and dried with filter paper, and quickly placed in liquid nitrogen for cold storage; according to the steps of the kit instruction manual, the OD value is measured, the standard curve is drawn according to the OD value of the known concentration of the standard substance, and the content of 5-HT, NE and GABA in the hippocampus of each group of mice is calculated according to the standard curve;

[0090] Antidepressant model evaluation seven:

[0091] Golgi staining: Golgi staining was used to observe the synaptic plasticity of the mouse hippocampus. Mouse brain tissue was immersed in Golgi staining solution for 48 hours, then the staining solution was changed once, and then changed every 3 days for a total of 14 days, stored at room temperature in the dark. Next, the brain tissue was washed three times with distilled water, then immersed in 80% glacial acetic acid overnight. After the tissue softened, it was washed with distilled water and placed in 30% sucrose. Then, the tissue was cut into 100 μm thick sections and mounted 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, dried, and sealed with glycerol gelatin. Photographs were taken using a Nikon DS-U3. The dendrites and dendritic spines in the hippocampus were analyzed using ImageJ software.

[0092] Example 7: Evaluation of the antidepressant activity of desvenlafaxine-γ-aminobutyric acid (GABA)

[0093] ICR mice (Experimental Animal Quality Certificate No.: NO.202370789), male, 22-30g, were randomly divided into control group, model group, norvenlafaxine 16.78mg / kg group (ODV 16.78mg / kg), γ-aminobutyric acid 6.57mg / kg group (GABA 6.57mg / kg), and norvenlafaxine-γ-aminobutyric acid 34.32mg / kg group (ODV-GABA 34.32mg / kg), with n=10 in each group. The control group and model group were injected intraperitoneally with the corresponding dose of physiological saline, while the drug treatment group was given the corresponding dose of drug intraperitoneally, with an administration volume of 10ml / kg.

[0094] Experimental results:

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

[0096] After 28 days of modeling, the mice underwent behavioral evaluation; such as... Figure 4 As shown in Figure A, the mean body weights of mice in the control group, model group, norvenlafaxine 16.78 mg / kg group, γ-aminobutyric acid (GABA) 6.57 mg / kg group, and norvenlafaxine-γ-aminobutyric acid (NABA) 34.32 mg / kg group were 35.09 g, 30.29 g, 33.18 g, 32.74 g, and 34.42 g, respectively. Compared with the model group, the body weights of mice in the drug treatment groups increased by 8.7%, 8.1%, and 13.63% at 28 days, respectively, with the norvenlafaxine-γ-aminobutyric acid group showing the greatest increase in body weight. Figure 4As shown in B, the sucrose preference of the control group, the model group, the nortriptyline 16.78 mg / kg group, the gamma-aminobutyric acid 6.57 mg / kg group, and the nortriptyline-gamma-aminobutyric acid 34.32 mg / kg group were 76.70%, 57.21%, 73.13%, 69.72%, and 76.17% respectively within 12 hours; the sucrose preference of the mice in the drug treatment groups was obviously improved compared with the model group; among them, the sucrose preference of the mice in the nortriptyline-gamma-aminobutyric acid group was obviously higher than that in the two single-drug groups; as shown in Figure 4 As shown in C-D, in the 6-minute tail suspension test and the forced swimming test, the immobility time of the control group, the model group, the nortriptyline 16.78 mg / kg group, the gamma-aminobutyric acid 6.57 mg / kg group, and the nortriptyline-gamma-aminobutyric acid 34.32 mg / kg group 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, and 74.8 s / 110.32 s respectively after 4 minutes; the immobility time of the mice in the model group was greatly increased compared with the control group, indicating that the chronic unpredictable mild stress modeling could cause the mice to have a reduced survival instinct and to produce behavioral despair, but the immobility time of the mice decreased after drug treatment, indicating that the survival instinct of the mice was improved after drug treatment;

[0097] The open field test is to explore the curiosity, exploration ability and anxiety of the mice; as shown in Figure 4 As shown in E, the total moving distance of the mice within 5 minutes in the control group, the model group, the nortriptyline 16.78 mg / kg group, the gamma-aminobutyric acid 6.57 mg / kg group, and the nortriptyline-gamma-aminobutyric acid 34.32 mg / kg group were 9.69 m, 5.65 m, 8.56 m, 7.95 m, and 9.06 m respectively; the total moving distance of the mice in the drug treatment groups was increased by 51.50%, 41.24%, and 60.35% respectively compared with the model group, and the nortriptyline-gamma-aminobutyric acid had the largest increase in the total moving distance; as shown in Figure 4 As shown in F, it can be seen from the mouse movement trajectory diagram that the movement trajectory of the mice in the model group was reduced compared with the control group, but the movement activity of the mice was obviously improved after drug treatment; as shown in Figure 4 As shown in G, the total moving time of the mice within 5 minutes in the control group, the model group, and each drug treatment group were 122.62 s, 72.29 s, 118.74 s, 117.33 s, and 123.06 s respectively; according to the data, the total moving time of the mice was the longest when treated with nortriptyline-gamma-aminobutyric acid, followed by the nortriptyline single-drug and the gamma-aminobutyric acid single-drug treatment; as shown in Figure 4As shown in H, the residence time of the control group, the model group and each drug administration group in the center area was 31.41 s, 15.81 s, 20.36 s, 21.4 s and 24.05 s, respectively. The residence time of the mice in the center area was the longest in the norvenlafaxine-gamma-aminobutyric acid group, followed by the gamma-aminobutyric acid group and the norvenlafaxine group. This indicated that the drug treatment strengthened the exploration desire of the mice, and the effect of norvenlafaxine-gamma-aminobutyric acid was the most significant;

[0098] In summary, in the behavioral evaluation, chronic unpredictable mild stress caused the weight loss of the mice, the decrease of sucrose preference, the increase of the immobility time in the tail suspension test and the forced swimming test, the decrease of the total moving distance, the decrease of the total moving time and the decrease of the residence time in the center area. However, after the treatment of norvenlafaxine, gamma-aminobutyric acid or norvenlafaxine-gamma-aminobutyric acid, the weight of the mice increased, the sucrose preference increased, the immobility time in the tail suspension test and the forced swimming test decreased, the total moving distance increased, the total moving time increased and the residence time in the center area increased. This indicated that the pleasure, the despair behavior, the curiosity and the exploration desire of the mice were improved, and the anxiety was relieved. The norvenlafaxine-gamma-aminobutyric acid combination had obvious advantages over the single drug group in the above behavioral evaluation. Therefore, the norvenlafaxine-gamma-aminobutyric acid combination had good antidepressant and anxiolytic effects.

[0099] (2) Effect of norvenlafaxine-gamma-aminobutyric acid combination on neurotransmitters in the brain of chronic unpredictable mild stress mice

[0100] Studies have shown that the increase of the level of monoamine neurotransmitters is related to the antidepressant effect. In order to study the anxiolytic and antidepressant effects of venlafaxine and gamma-aminobutyric acid, the contents of 5-HT, NE and GABA in the hippocampus of the mice were detected by Elisa. Figure 5 As shown in A, the contents of 5-HT in the hippocampus of the control group, the model group and each drug administration group were 46.27 ng / mg, 25.45 ng / mg, 44.64 ng / mg, 40.59 ng / mg and 46.27 ng / mg, respectively. Figure 5 As shown in B, the contents of NE were 166.17 pg / mg, 97.5 pg / mg, 146 pg / mg, 125.51 pg / mg and 158.06 pg / mg, respectively. Figure 5 As shown in C, the contents of GABA were 12.27 pg / mg, 5.33 pg / mg, 7.55 pg / mg, 7.68 pg / mg and 10.38 pg / mg, respectively. When the drugs were administered, the contents of 5-HT, NE and GABA in the mice treated with the norvenlafaxine-gamma-aminobutyric acid combination were higher than those in the mice treated with norvenlafaxine or gamma-aminobutyric acid.

[0101] In summary, chronic unpredictable mild stress leads to a decrease in the levels of 5-HT, NE, and GABA in the hippocampus of mice. Drug treatment increases the levels of 5-HT, NE, and GABA. Treatment with desvenlafaxine-γ-aminobutyric acid (DABA) significantly improves neurotransmitter levels compared to monotherapy, indicating that DABA treatment can significantly reverse neuronal damage induced by chronic unpredictable mild stress and increase neurotransmitter levels in the brain.

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

[0103] To determine whether chronic, unpredictable, mild stress induces damage to the synaptic structure of neurons in the CA1 region of the hippocampus, we performed Golgi staining on mouse brain slices, such as... Figure 6 As shown in Figures AB, density analysis of single dendritic spines using ImageJ software revealed that the dendritic spine density in the CA1 region of the hippocampus of the control group mice was 9.93 / 10 μm, while the dendritic spine density in the CA1 region of the model group mice was 5.48 / 10 μm. After drug treatment, the dendritic spine density in the CA1 region of the hippocampus of mice treated with norvenlafaxine 16.78 mg / kg, γ-aminobutyric acid 6.57 mg / kg, and norvenlafaxine-γ-aminobutyric acid was 7.93 / 10 μm, 5.85 / 10 μm, and 5.85 / 10 μm, respectively, compared to the model group. The density of hippocampal neurons in the CA1 region of mice in the drug treatment group was 44.7%, 0.67%, and 68.43% higher than that in the model group, at 0 μm and 9.23 / 10 μm, respectively. The data showed that the increase in neuronal density was most significant after treatment with desvenlafaxine-γ-aminobutyric acid (GABA), indicating that desvenlafaxine treatment is more effective than single-drug treatment in restoring neuronal damage. In addition, path tracing of individual neuronal branches on Golgi-stained brain slices was performed, and the ShollAnalysis plugin was used for analysis and statistical analysis. Figure 6 CD results showed that, compared with the control group, the number of neuronal intersections in the CA1 region of the hippocampus in the model group was significantly higher in the range of 0-100 μm, with the highest number of intersections at 50 μm. 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 indicated that chronic unpredictable mild stress modeling reduces the dendritic complexity of neurons in the hippocampus. When desvenlafaxine or γ-aminobutyric acid (GABA) monotherapy was administered, the dendritic complexity of neurons in the CA1 region of the hippocampus increased. However, the dendritic complexity of neurons in the CA1 region of the hippocampus was significantly better in the desvenlafaxine-γ-aminobutyric acid monotherapy group than in the monotherapy group.

[0104] In summary, chronic unpredictable mild stress can cause the decrease of neuron density and the decrease of neuron dendritic complexity in the hippocampal CA1 region of mice, and the neuron density and the neuron dendritic complexity in the hippocampal CA1 region of mice are increased after drug treatment, among which the neuron damage of mice is the least after the treatment of ODV-GABA combination, indicating that the effect of ODV-GABA combination on neuron repair is more significant than that of single drug treatment.

[0105] Example 8: Evaluation of antidepressant activity of ODV and GABA combination

[0106] ICR mice (experimental animal quality certificate number: NO.202370789), male, 22-30 g, were randomly divided into control group, model group, ODV 16.78 mg / kg group (ODV 16.78 mg / kg), GABA 6.57 mg / kg group (GABA 6.57 mg / kg), ODV and GABA group (ODV 16.78 mg / kg+GABA 6.57 mg / kg), n=10 in each group; the control group and the model group were injected with the corresponding dose of normal saline, and the drug treatment groups were given the corresponding dose of drugs by intraperitoneal injection, and the volume of drug administration was 10 ml / kg;

[0107] Experimental results

[0108] (1) Effect of ODV and GABA combination on the behavior of mice with chronic unpredictable mild stress

[0109] Behavioral evaluation of mice, as shown in Figure 7 A, the average body weight of mice in the control group, the model group, the ODV 16.78 mg / kg group, the GABA 6.57 mg / kg group, and the ODV 16.78 mg / kg and GABA 6.57 mg / kg group was 35.09 g, 30.29 g, 33.18 g, 32.74 g, and 33.64 g, respectively; the body weight of mice in the drug treatment groups increased by 8.7%, 8.1%, and 9.96% compared with the model group, among which the body weight of mice increased the most in the ODV and GABA combination treatment group; as shown in Figure 7 B, the sucrose preference of the control group, the model group, the ODV 16.78 mg / kg group, the GABA 6.57 mg / kg group, and the ODV and GABA combination was 76.70%, 57.21%, 73.13%, 69.72%, and 74.84%, respectively, among which the sucrose preference value of the combination increased the most compared with the model group, indicating that the hedonic sensation of mice was improved, followed by the ODV and GABA group; as shown in Figure 7As shown in C-D, in the tail suspension test and forced swimming test, the normal group, model group, nortriptyline 16.78 mg / kg group, gamma-aminobutyric acid 6.57 mg / kg group and nortriptyline and gamma-aminobutyric acid composition group, the rest time of 4 min is 110.32 s / 107.53 s, 143.06 s / 153.22 s, 105.02 s / 116.13 s, 120.32 s / 141.21 s, 89.91 s / 113.68 s, respectively. Compared with the model group, the rest time of the drug treatment group in the tail suspension test and forced swimming test was reduced by 26.60% / 24.21%, 15.90% / 7.8%, 37.15% / 25.81%, respectively. The experimental results show that after giving drug treatment, the rest time of nortriptyline and gamma-aminobutyric acid composition is decreased most obviously, which indicates that the composition treatment better relieves the despair behavior of mice;

[0110] As shown in Figure 7 As shown in E, in the open field test, the total moving distance of mice for 5 min, the normal group, model group, nortriptyline 16.78 mg / kg group, gamma-aminobutyric acid 6.57 mg / kg group and nortriptyline and gamma-aminobutyric acid composition group, the total moving distance is 9.69 m, 5.65 m, 8.56 m, 7.95 m, 8.74 m, respectively. Compared with the model group, the total moving distance of mice in the drug treatment group was increased by 51.5%, 40.5%, 54.7%, respectively. The total moving distance of the composition is increased most, which indicates that the activity of mice is strengthened; as shown in Figure 7 F, the mouse movement trajectory diagram, the movement trajectory of the model group mice is reduced compared with the control group, but after single drug or composition treatment, the movement activity of mice is obviously improved; as shown in Figure 7 G, the total moving time of mice in the control, model and each drug group is 122.62 s, 72.29 s, 118.74 s, 117.33 s, 121.68 s, respectively. The total moving time of mice in the nortriptyline and gamma-aminobutyric acid composition group is higher than that in the nortriptyline single drug and gamma-aminobutyric acid single drug; as shown in Figure 7 H, the residence time of mice in the center area of the control, model and each drug group is 31.41 s, 15.81 s, 20.36 s, 21.4 s, 22.51 s, respectively. Among them, the residence time of mice in the center area treated by nortriptyline and gamma-aminobutyric acid composition is the most, followed by gamma-aminobutyric acid single drug treatment, which indicates that the exploration desire of mice is improved and the anxiety is relieved after composition treatment;

[0111] In summary, in the behavioral evaluation, chronic unpredictable mild stress caused weight loss, sucrose preference decreased, the tail suspension test and forced swimming test in the static time longer, the total distance decreased, the total time decreased and the center area stay time decreased, but after treatment with norepinephrine, gamma-aminobutyric acid or combination of norepinephrine and gamma-aminobutyric acid composition, the mice increased weight, sucrose preference, tail suspension test and forced swimming test in the static time decreased, the total distance increased, the total time increased and the center area stay time increased; indicating that the mouse's pleasure, despair behavior, curiosity and exploration desire of the mouse are improved, and the anxiety is relieved; the drug combination treatment has obvious advantages in the above behavioral evaluation compared with single drug treatment, therefore, the combination of norepinephrine and gamma-aminobutyric acid has good antidepressant and anxiolytic effects;

[0112] (2) Effect of the combination of norepinephrine and gamma-aminobutyric acid on neurotransmitters in the brain of chronic unpredictable mild stress mice

[0113] As shown in Figure 8 A, the 5-HT content in the hippocampus of mice, the 5-HT content in the hippocampus of mice in the control group, the model group and each drug administration group was 46.27 ng / mg, 25.45 ng / mg, 44.64 ng / mg, 40.59 ng / mg and 47.63 ng / mg, respectively; the 5-HT content in the drug treatment groups was increased by 75.4%, 59.49% and 87.15% compared with the model group, respectively; the 5-HT content in the combination of norepinephrine and gamma-aminobutyric acid was the highest, followed by norepinephrine and gamma-aminobutyric acid; as shown in Figure 8 B, the NE content was 166.17 pg / mg, 97.5 pg / mg, 146 pg / mg, 125.51 pg / mg and 150.22 pg / mg, respectively; the NE content in the drug treatment groups was increased by 49.74%, 24.63% and 62.11% compared with the model group, respectively; the NE content in the combination of norepinephrine and gamma-aminobutyric acid was the highest, followed by norepinephrine and gamma-aminobutyric acid; as shown in Figure 8 C, the GABA content in the control group, the model group and each drug administration group was 12.27 pg / mg, 5.33 pg / mg, 7.55 pg / mg, 7.68 pg / mg and 9.71 pg / mg, respectively; the GABA content in the drug treatment groups was increased by 41.65%, 44.09% and 94.75% compared with the model group, respectively; the GABA content in the combination of norepinephrine and gamma-aminobutyric acid was higher than that in the single drug treatment groups;

[0114] In summary, CUMS can cause the content of 5-HT, NE and GABA in the hippocampal CA1 region of mice to decrease, and after drug treatment, the content of 5-HT, NE and GABA neurotransmitters in the composition is higher than that in the single drug group, and the content of nortriptyline neurotransmitter is higher than that of 5-HT and NE, but in the GABA index, the content of the γ-aminobutyric acid group is slightly higher than that of the nortriptyline group; The data shows that the nortriptyline and γ-aminobutyric acid composition has a better advantage in improving the content of neurotransmitters in the brain than the single drug, so the composition has a significant advantage in the treatment of depression;

[0115] (3) Effect of nortriptyline and γ-aminobutyric acid composition on synaptic structural plasticity of hippocampal CA1 neurons of chronic unpredictable mild stress mice

[0116] As shown in Figure 9 A-B, ImageJ software was used to analyze the density of single dendritic spine, and it was found that the dendritic spine density of the control group, the model group and each drug group in the hippocampal CA1 region of mice was 9.93 / 10 μm, 5.48 / 10 μm, 7.93 / 10 μm, 5.85 / 10 μm and 8.48 / 10 μm, respectively. The neuron density of the drug group increased by 44.7%, 0.67% and 54.74% compared with the model group, respectively. It can be concluded that the neuron density of the mice increases most obviously when treated with the composition; In addition, the ShollAnalysis plug-in was used to track the path of single neuron branches on the brain slices after Golgi staining, as shown in Figure 9 C-D results show that the number of intersections is the highest at 50 μm, and the number of intersections of the control group, the model group and each drug group is 17, 5, 11, 6 and 13, respectively. It can be concluded that the number of intersections of the composition is the highest in each drug group, so the recovery effect of the composition on neuron damage is better than that of the single drug;

[0117] In summary, chronic unpredictable mild stress can cause the neuron density in the hippocampal CA1 region of mice to decrease, and the neuron density in the hippocampal CA1 region of mice to increase after drug treatment; Among them, the neuron density increases the most after treatment with the composition, and the complexity is higher, indicating that the nortriptyline and γ-aminobutyric acid composition treatment has a more significant effect on neuron repair than the single drug treatment.

[0118] Example 9, Evaluation of the antidepressant activity of the nortriptyline and γ-aminobutyric acid composition

[0119] ICR mice (experimental animal quality certificate number: NO. 202370789), male, 22-30 g, the animals were randomly divided into control group, model group, venlafaxine 20 mg / kg group (VLF 20 mg / kg), gamma-aminobutyric acid 7 mg / kg group (GABA 7 mg / kg), venlafaxine and gamma-aminobutyric acid composition group (VLF 20 mg / kg+GABA 7 mg / kg), n=11 in each group; the control group and the model group were injected intraperitoneally with the corresponding dose of normal saline, and the drug treatment group was given the corresponding dose of drug intraperitoneally, and the drug volume was 10 ml / kg.

[0120] Experimental results

[0121] (1), the effect of venlafaxine and gamma-aminobutyric acid composition on the behavior of chronic unpredictable mild stress mice

[0122] As Figure 10 A shows that the average body weight of mice in the control group, model group, venlafaxine 20 mg / kg group, gamma-aminobutyric acid 7 mg / kg group, and venlafaxine and gamma-aminobutyric acid composition group is 32.15 g, 28.47 g, 31.29 g, 30.79 g, and 31.7 g, respectively; compared with the model group, the body weight of each drug treatment group increased by 9.9%, 8.15%, and 11.34%, respectively, and the body weight of mice after treatment with the composition increased more obviously; as Figure 10 B shows that the sucrose preference of the normal group, model group, and drug treatment group is 84.84%, 61.06%, 74.08%, 70.36%, and 74.62%, respectively; as can be seen from the data, the sucrose preference of the composition is higher than that of venlafaxine and gamma-aminobutyric acid alone; as Figure 10 C shows that in the tail suspension test, the resting time of mice in the normal group, model group, and drug treatment group after 4 min is 104.78 s, 134 s, 106.45 s, 120.1 s, and 98.25 s, respectively; the resting time of mice treated with venlafaxine and gamma-aminobutyric acid composition is the shortest among the drug treatments, indicating that the behavior despair of mice is better relieved; as Figure 10 D shows that in the forced swimming test, the resting time of mice in the normal group, model group, and drug treatment group after 4 min is 110.69 s, 143.96 s, 107.66 s, 127.17 s, and 95.89 s, respectively; as can be seen from the data, the resting time of mice treated with venlafaxine and gamma-aminobutyric acid alone or composition is decreased in the forced swimming test, indicating that the survival desire of mice is enhanced, and the resting time of mice treated with the composition is the lowest, indicating that the treatment effect of the composition is better than that of single drug treatment;

[0123] As Figure 10E shows that the total distance of movement of mice in the open field experiment for 5 min, the total distance of movement of the control group, the model group, the venlafaxine 20 mg / kg group, the gamma-aminobutyric acid 7 mg / kg group, and the combination of venlafaxine and gamma-aminobutyric acid group are 14.16 m, 7.24 m, 11.07 m, 10.62 m, and 11.47 m, respectively, wherein the total distance of movement of mice in each drug treatment group is increased by 52.90%, 46.69%, and 58.43% compared with the model group; the total distance of movement of mice in the combination of venlafaxine and gamma-aminobutyric acid group is the highest, indicating that the motor activity of mice in the combination treatment group is the best; as shown in Figure 10 F shows the mouse movement trajectory diagram, the movement trajectory of the model group is reduced compared with the control group, but the motor activity of mice in the drug treatment group is obviously improved; as shown in Figure 10 G shows that the total movement time of mice in the control group, the model group, and each drug treatment group are 166.21 s, 96.14 s, 133.68 s, 136.69 s, and 142.89 s, respectively, and the total movement time of mice in the combination of venlafaxine and gamma-aminobutyric acid group is obviously higher than that in the single drug group; as shown in Figure 10 H shows that the central zone residence time of mice in the control group, the model group, and each drug treatment group are 23.14 s, 15.56 s, 24.28 s, 28.04 s, and 24.9 s, respectively, and the central zone residence time of mice in the combination of venlafaxine and gamma-aminobutyric acid group is the longest, indicating that the exploration desire of mice in the combination treatment group is the strongest;

[0124] In summary, in the behavioral evaluation, chronic unpredictable mild stress causes weight loss, decreased sucrose preference, increased immobility time, decreased total distance of movement, decreased total movement time, and decreased central zone residence time in the tail suspension test and forced swimming test, but after treatment with venlafaxine, gamma-aminobutyric acid, or the combination of venlafaxine and gamma-aminobutyric acid, the weight of mice increases, the sucrose preference improves, the immobility time decreases, the total distance of movement increases, the total movement time increases, and the central zone residence time increases; the above behaviors indicate that the hedonic effect, despair behavior, curiosity, and exploration desire of mice are improved and the anxiety is alleviated; the combination of venlafaxine and gamma-aminobutyric acid has obvious advantages in the behavioral evaluation compared with the single drug, therefore, the combination of venlafaxine and gamma-aminobutyric acid has good antidepressant and anxiolytic effects;

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

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

[0127] In summary, chronic unpredictable mild stress can reduce the levels of 5-HT, NE, and GABA in the hippocampus of mice. When treated with drugs, the levels of neurotransmitters in the combination drug were higher than those in the single drug group in terms of 5-HT, NE, and GABA, and venlafaxine was higher than that in γ-aminobutyric acid. The results indicate that both single drug and combination drug treatments can increase the levels of neurotransmitters in the brain, but the combination drug treatment is more effective.

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

[0129] like Figure 12 As shown in Figures AB, ImageJ software density analysis of single dendritic spines revealed that the dendritic spine densities in the CA1 region of the mouse hippocampus were 7.13 / 10μm, 3.39 / 10μm, 5.8 / 10μm, 4.44 / 10μm, and 6.15 / 10μm in the control, model, and drug-treated groups, respectively. Compared to the model group, the neuronal density in the drug-treated groups increased by 41.56%, 30.97%, and 81.42%, respectively. Furthermore, the Sholl Analysis plugin was used to trace the paths of individual neuronal branches on brain slices stained with Golgi apparatus, such as...Figure 11 The C-D results show that the number of neuron intersection points in the control group is less than that in the model group within 0-100 μm, but after drug treatment, the number of neuron intersection points in the hippocampal CA1 region of mice increases, and the number of intersection points in the composition treatment group is higher than that in the single drug treatment group, which also indicates that the composition enhances the complexity of neuron dendrites;

[0130] In summary, CUMS can cause the decrease of neuron density and the decrease of neuron dendritic complexity in the hippocampal CA1 region of mice, and after drug treatment, the neuron density in the hippocampal CA1 region of mice increases and the neuron dendritic complexity improves; among them, the neuron density in the hippocampal CA1 region of mice increases the most after treatment with the combination of venlafaxine and gamma-aminobutyric acid composition, and the neuron dendritic complexity is higher, indicating that the composition treatment has better effect on neuron repair than the single drug treatment.

Claims

1. A class of conjugate drugs having synergistic antidepressant effects, characterized in that, The conjugate is a compound or its pharmaceutically acceptable salt formed by condensation of nortriptyline and gamma-aminobutyric acid, and its chemical structural formula is shown as formula I:

2. The method of claim 1, wherein the preparation of the class of drugs having synergistic anti-depressant effect is characterized by, The preparation steps are as follows: Step (1): protecting the amino group in the structure of gamma-aminobutyric acid by di-tert-butyl dicarbonate or benzyl chloroformate to obtain intermediate I; Step (2): condensing nortriptyline and the intermediate I obtained in step (1) under EDCI / DMAP to obtain 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 conjugate; The specific synthesis route is as follows: 。 3. A conjugate composition comprising the compound or its pharmaceutically acceptable salt formed by condensation of nortriptyline and gamma-aminobutyric acid according to claim 1 and a pharmaceutically acceptable carrier.

4. Use of the compound or its pharmaceutically acceptable salt formed by condensation of nortriptyline and gamma-aminobutyric acid according to claim 1 and the conjugate composition according to claim 3 in the preparation of a medicament for preventing or treating depression.

5. Use according to claim 4, characterized in that: The depression is major depression, bipolar depression, psychotic depression, reactive depression, menopausal depression, childhood depression, secondary depression, postpartum depression, seasonal depression, persistent depression, postmenopausal and perimenopausal depression, anxiety depression and panic disorder.

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