Medical use of an aporphine alkaloid

Abstract

The application discloses a use of an aporphine alkaloid shown in formula III in preparation of an antidepressant, wherein R1 and R2 are independently selected from alkoxy and methylenedioxy, R3 and R4 are independently selected from H, OH, alkoxy and methylenedioxy, but R3 and R4 cannot be H at the same time or R1 and R2 are both selected from methoxy, and R3 cannot be OH and R4 cannot be H. Pharmacological experiments show that the aporphine alkaloid shown in formula III has better antidepressant effect, higher response rate and faster effect than fluoxetine.

Description

[0001] This application is a divisional application of the invention entitled "Medical Use of Apophyte Alkaloids" filed on March 5, 2021, with application number 2021102449157. Technical Field

[0002] This invention belongs to the field of pharmacotherapeutics and relates to the pharmaceutical use of apophysine alkaloids, specifically the use of apophysine alkaloids in antidepressant drugs. Background Technology

[0003] Depression is a mental disorder characterized by a persistent depressed mood as its primary clinical symptom. Clinical manifestations include depressed mood, anxiety, lethargy, and numerous physical symptoms, loss of interest in pleasurable activities, feelings of guilt or worthlessness, and in severe cases, suicidal ideation and behavior. Sometimes, accompanying symptoms may occur, such as decreased energy, changes in appetite, more or less sleep, anxiety, decreased attention, and agitation. The etiology and pathogenesis of depression are not yet fully understood. Low levels of monoamine neurotransmitters such as norepinephrine, serotonin, and dopamine in the central nervous system, as well as impaired receptor function, are considered contributing factors to depression.

[0004] Existing clinical antidepressants have drawbacks such as slow onset of action, low response rate, and side effects (such as withdrawal effects and gastrointestinal symptoms, including dry mouth, constipation, and diarrhea). Summary of the Invention

[0005] The purpose of this invention is to address the problems of existing clinical antidepressants, such as slow onset of action, low response rate, and side effects (such as withdrawal effects, gastrointestinal symptoms, dry mouth, constipation, and diarrhea after administration), and to provide an antidepressant pharmaceutical use of apophyte alkaloids. Pharmacological experiments show that apophyte alkaloids represented by Formula III have better antidepressant efficacy and, compared with fluoxetine, have better antidepressant effects, higher response rate, and faster onset of action.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] The use of apophyll alkaloids as shown in Formula III in the preparation of antidepressant drugs;

[0008]

[0009] Among them, R1 and R2 are independently selected from alkoxy and methylenedioxy (-OCH2O-), respectively, and R3 and R4 are independently selected from H, OH, alkoxy and methylenedioxy, respectively. However, R3 and R4 cannot be H at the same time, or when R1 and R2 are selected from methoxy, R3 cannot be OH and R4 cannot be H.

[0010] The 9th and 10th positions of apophyne alkaloids are both active sites, with the alkoxy group at position 10 exhibiting the best activity. Furthermore, when 1,2-substituted with methylenedioxy or simultaneously substituted with methoxy, the antidepressant activity of apophyne alkaloids with single or double substitution at positions 9 or 10 is improved compared to apophyne alkaloids without substitution at positions 9 and 10.

[0011] Preferably, R1 and R2 are independently selected from alkoxy and methylenedioxy, respectively, and R3 and R4 are independently selected from H, OH, alkoxy, and methylenedioxy, respectively. However, R3 and R4 cannot be H at the same time, or R1 and R2 are both selected from methoxy, in which case R3 cannot be OH and R4 cannot be H, or R1 and R2 are selected from methylenedioxy, in which case R3 cannot be OH and R4 cannot be H or methoxy, or R1 and R2 are selected from methylenedioxy, in which case R3 cannot be methoxy and R4 cannot be OH, or R1 and R2 are selected from methylenedioxy, in which case R3 cannot be H and R4 cannot be methoxy.

[0012] More preferably, R1 and R2 are independently selected from alkoxy and methylenedioxy, respectively, R3 is selected from methoxy, and R4 is selected from H, or R1 and R2 are both selected from methoxy, R3 is selected from OH, and R4 is selected from methoxy, or R1 and R2 are selected from methylenedioxy, R3 is selected from methoxy, and R4 is selected from methoxy.

[0013] The alkoxy group described in this invention is selected from C1 to C5 alkoxy groups, specifically from methoxy, ethoxy, and C3 to C5 straight-chain or branched alkoxy groups.

[0014] Specifically, the apophene alkaloids represented by Formula III are preferably selected from the following compounds:

[0015] Another object of the present invention is to provide a pharmaceutical composition having apophyseal alkaloids of Formula III as the active ingredient, supplemented with a pharmaceutically acceptable carrier, and formulated into any pharmaceutically acceptable dosage form.

[0016] The dosage form is selected from tablets, capsules, pellets, granules, powders, lozenges, aqueous or oily suspensions, and injections.

[0017] Another object of the present invention is to provide the use of the pharmaceutical composition in the preparation of a therapeutic antidepressant. Attached Figure Description

[0018] Figure 1 Effects of Series I apophytic alkaloids on the locomotion (total distance traveled) of zebrafish.

[0019] Figure 2 Effects of Series I apophytic alkaloids on the locomotion (swimming speed) of zebrafish.

[0020] Figure 3Effects of Series IV apophytic alkaloids on the locomotion (total distance traveled) of zebrafish.

[0021] Figure 4 Effects of Series IV apophytic alkaloids on the locomotion (swimming speed) of zebrafish.

[0022] Figure 5 Effects of Ie and fluoxetine on the movement trajectory of zebrafish, a depression model.

[0023] Figure 6 The effects of Ie and fluoxetine on the locomotion of zebrafish include: A. total swimming distance, B. average speed, C. time at rest, D. frequency at rest, E. swerving degree, F. turning angle, and G. angular velocity.

[0024] Figure 7 : Changes in mouse body weight; where A represents changes in mouse body weight after modeling, B represents changes in mouse body weight before and after drug administration, and C represents daily changes in mouse body weight during modeling and drug administration.

[0025] Figure 8 Effects of Ie on the open field behavior of reserpine-induced depression model C57BL / 6 mice; where A is total swimming distance, B is average speed, C is immobility time, D is immobility frequency, E is meandering degree, F is turning angle, and G is angular velocity.

[0026] Figure 9 Cluster analysis of mouse open field experimental behavior; where A represents cluster analysis of 50 biological samples and B represents cluster analysis of five experimental groups.

[0027] Figure 10 : Number of mouse fecal pellets in open field experiment.

[0028] Figure 11 Number of times the mouse stood upright in the open field experiment.

[0029] Figure 12 Effect of compound Ie on immobility time in the tail suspension test of C57BL / 6 mice with reserpine-induced depression.

[0030] Figure 13 Effect of compound Ie on immobility time in the forced swimming test of reserpine-induced depression model C57BL / 6 mice.

[0031] Figure 14 The response rate and cure rate of compound Ie in reserpine-induced depression model C57BL / 6 mice. Detailed Implementation

[0032] The technical solution of the present invention will be further described in conjunction with specific embodiments.

[0033]

[0034] Table 1. Alphaphene-like alkaloids of the present invention

[0035]

[0036]

[0037] Example 1: In vivo pharmacological experiment of Series I apophytic alkaloids in zebrafish

[0038] The antidepressant activity of reserpine was tested using an open field (OFT) experiment and a zebrafish depression model, with fluoxetine (Flu) selected as a positive control.

[0039] Instrument: Zebrafish Behavioral Analyzer

[0040] Reagents: Compounds Ia, Ib, Ic, Id, Ie, Ig, Ii, and Ik (all known compounds); fluoxetine and reserpine (purity ≥99.0%) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0041] Zebrafish: Wild-type AB strain adult zebrafish, with broodstock provided by the Wuhan National Zebrafish Resource Center and bred in the zebrafish laboratory of Nanjing Ruiying Runze Biomedical Technology Co., Ltd. Zebrafish were raised according to The Zebrafish Book, in recirculating water, with water quality maintained through the recirculating water system, and the water temperature kept at 28.5℃. The daily light and dark time was fixed at 14h / 10h, and brine shrimp were fed twice a day, morning and evening. The stocking density was no more than 5 adult fish per 1L of aquaculture tank.

[0042] Methods: Modeling and grouping of depressed zebrafish: Zebrafish were randomly divided into 11 groups: blank control group (10 zebrafish), model group (10 zebrafish), positive control group (10 zebrafish, fluoxetine 1 μM), and treatment groups: Ia group (10 zebrafish, 1 μM), Ib group (10 zebrafish, 1 μM), Ic group (10 zebrafish, 1 μM), Id group (10 zebrafish, 1 μM), Ie group (10 zebrafish, 1 μM), Ig group (10 zebrafish, 1 μM), Ii group (10 zebrafish, 1 μM), and Ik group (10 zebrafish, 1 μM). Adult zebrafish (six months old) were subjected to acute stress in a 10 mg / L reserpine solution for 40 minutes, then removed and cultured in 2 L beakers with drug-free water. After 24 hours, the positive control group was given 1 μM fluoxetine (fluoxetine was prepared as a 10 mmol / L stock solution with physiological saline, then diluted to a final concentration of 1 μM with the culture water). The drug treatment group received 1 μM of each of the compounds Ia, Ib, Ic, Id, Ie, Ig, Ii, and Ik (compounds were prepared as 10 mmol / L stock solutions with DMSO, then diluted to a final concentration of 1 μM with the culture water). The blank control group and the model group received the same volume of drug-free water. During the drug treatment period, the culture water containing the drug concentration in the drug treatment group was changed daily, while the culture water in the blank control group and the model group was changed to drug-free water. Behavioral tests were performed on the zebrafish in each group after 7 days. Behavioral verification was conducted using an open field experiment. Zebrafish were placed in a 2L container of water (the container dimensions were 30×30×10cm (length×width×height), with a white frosted bottom and black frosted sides, made of acrylic sheet). A camera was placed 45cm above the center of the top of the tank. Without needing to adapt to the environment, the movement curves of the zebrafish over 5 minutes were recorded using the Nordas EthoVision XT 11.5 animal movement trajectory tracking system, along with the following variables: A. total swimming distance; B. average speed.

[0043] Result: As Figure 1 and Figure 2 As shown, compared with the blank control group (Control), the total distance traveled and the swimming speed of the zebrafish in the model group (Modle) were significantly reduced. #### P<0.0001, indicating that the reserpine-induced depression model was successfully established in zebrafish. Compared with the model group, the reserpine-treated groups (Ia, Ib, Ic, Id, Ie, Ig) and the positive control group all showed significant increases in zebrafish exploratory behavior, total swimming distance, and swimming speed. **** P<0.0001, *** P<0.001, ** P<0.01, * (P<0.05) Behavioral indicators showed that the Ie group had the best effect and showed better antidepressant efficacy than the positive control group.

[0044] Example 2: In vivo pharmacological experiment of series IV apophytic alkaloids in zebrafish

[0045] Reagents: Compounds IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVi, and IVk (all known compounds).

[0046] Zebrafish were randomly divided into 12 groups: a blank control group (10 zebrafish), a model group (10 zebrafish), a positive control group (10 zebrafish, 1 μM fluoxetine), and treatment groups: IVa group (10 zebrafish, 1 μM), IVb group (10 zebrafish, 1 μM), IVc group (10 zebrafish, 1 μM), IVd group (10 zebrafish, 1 μM), IVe group (10 zebrafish, 1 μM), IVf group (10 zebrafish, 1 μM), IVg group (10 zebrafish, 1 μM), IVVi group (10 zebrafish, 1 μM), IVk group (10 zebrafish, 1 μM), and the rest were the same as in Example 1.

[0047] Result: As Figure 3 and Figure 4 As shown, compared with the blank control group, the total distance traveled and the swimming speed of zebrafish in the model group were significantly reduced. #### P<0.0001, indicating that the reserpine-induced depression model was successfully established in zebrafish. Compared with the model group, the IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVVi groups and the positive control group all showed significant increases in zebrafish exploratory behavior, total swimming distance, and swimming speed. **** P<0.0001, *** P<0.001, ** P<0.01, * P<0.05).

[0048] Example 3: In vivo pharmacological experiment of Ie in zebrafish

[0049]

[0050] Zebrafish were randomly divided into four groups: a blank control group (10 zebrafish), a model group (10 zebrafish), a positive control group (10 zebrafish, fluoxetine 1 μM), and an Ie group (10 zebrafish, 1 μM). An animal motion tracking system was used to record the following variables on the zebrafish's movement curves over 5 minutes: A. Total swimming distance; B. Average speed; C. Time at which the zebrafish remained stationary; D. Frequency of stationary movement; E. Twist angle; F. Turning angle; G. Angular velocity. The rest was the same as in Example 1.

[0051] Result: As Figure 5 and Figure 6 As shown, compared with the blank control group, the total distance traveled and the swimming speed of the zebrafish in the model group were significantly reduced. ####P<0.0001), stationary time, stationary frequency, tortuosity, turning angle, and angular velocity increased significantly. #### P<0.0001, indicating that the reserpine-induced depression model was successfully established in zebrafish. Compared with the model group, both the positive control group and the Ie group significantly increased zebrafish exploratory behavior, total swimming distance, swimming speed, immobility time, meandering degree, turning angle, and angular velocity, thus improving the depressive state. **** P<0.0001, **** (P<0.001), and all behavioral indicators showed that compound Ie had the best effect, exhibiting better antidepressant efficacy than fluoxetine.

[0052] Example 4: In vivo pharmacological experiment of Ie in C57BL / 6 mice

[0053] The antidepressant activity of reserpine was tested in mice with a reserpine-induced depression model using the open field test (OFT), tail suspension test (TST), and forced swimming test (FST), with fluoxetine (Flu) selected as a positive control.

[0054] Instrument: ZS Behavioral Analysis Instrument from Beijing Zhongshidichuang Technology Co., Ltd.

[0055] Reagents: Compound Ie, fluoxetine, and reserpine (purity ≥99.0%) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0056] Materials: C57BL / 6 mice, male, weighing 21±2g.

[0057] method:

[0058] (1) Modeling and grouping of depressed mice: The test animals were kept under alternating light and dark conditions for 12 hours (lights on at 6:00 and off at 18:00), with a housing temperature of (25±2)℃ and humidity of 50%±20%. Water and bedding were changed regularly every 3 days, and the mice had free access to food and water. Five mice were kept per cage. After 7 days of acclimatization, the mice were randomly divided into 5 groups: blank control group (n=10), model group (n=10), positive control group (n=10, fluoxetine 20mg / kg), low-dose Ie group (n=10, 10mg / kg), and high-dose Ie group (n=10, 20mg / kg). The blank control group was injected with 0.2% acetic acid saline (acetic acid concentration is volume percentage); the other groups were injected intraperitoneally with reserpine 1mg / kg for 3 consecutive days to establish the depression model, and each group was administered the drug intraperitoneally at a dose of 10mL / kg. After modeling, the blank control group and the model group were injected with physiological saline, while the low-dose Ie group and the high-dose Ie group were administered the medication by gavage at a dose of 10 mL / kg once daily for one week. Reserpine was dissolved in 0.2% acetic acid saline, compound Ie was dissolved in 0.01% DMSO saline (DMSO concentration is volume percentage), and fluoxetine was dissolved in physiological saline to prepare concentrations sufficient for a final administration volume of 10 mL / kg for mice.

[0059] (2) Observation indicators

[0060] After the formal weight test begins, the mice are weighed daily, and the weight changes before and after the experiment are calculated.

[0061] One hour after the last drug administration, mice underwent Out-of-Field (OFT) testing. Before the test, animals were allowed to acclimatize in the testing room for one hour. Then, each animal was placed individually into a closed OFT box (50cm long, 50cm wide, and 50cm high). After a 1-minute acclimatization period, behavioral indicators such as total distance traveled, movement speed, immobility time, immobility frequency, angle, angular velocity, and zigzag degree of movement were recorded over 5 minutes. The number of times the mice stood upright and the number of fecal particles were also recorded during the test. After each animal's test, the OFT box was cleaned (wiped with 70% ethanol and allowed to dry) to avoid interference from the previous animal's scent.

[0062] One hour after the last administration of the C-tail suspension immobility test, mice underwent TST (Tail Suspension Test). Adhesive tape was fixed approximately 1 cm from the tip of the mouse's tail and suspended from the upper support of the instrument, allowing the mouse to hang upside down in the tail suspension box. The total test duration was 6 minutes. After 2 minutes of adaptation, the cumulative immobility time of the animals in the last 4 minutes was recorded (immobility means the mouse stopped struggling and remained still).

[0063] One hour after the last dose of the forced swimming immobility test, mice were placed in a cylindrical forced swimming container with a diameter of 10 cm, a height of 20 cm, and a water depth of 15 cm, and the water temperature was maintained at 25°C. After the animals adapted for 2 minutes, the computer image real-time detection and analysis system automatically recorded the cumulative immobility time of the animals in the following 4 minutes (mice that stopped struggling or floated on the water, with only slight movements to keep their heads above the water surface, were considered immobile).

[0064] Result: As Figure 7 A represents the change in mouse body weight after modeling. Compared with the blank control group, the body weight of mice in the model group decreased significantly. ## P<0.01 indicates that the reserpine-induced depression model in mice was successfully established. Figure 7 B represents the change in mouse body weight before and after drug administration. Compared with the model group, both the low-dose and high-dose Ie groups could restore the weight loss symptoms caused by depression in mice, with the high-dose Ie group showing the most significant effect. The difference was statistically significant. * (P<0.05), while the positive control group did not improve and aggravated the weight loss symptoms in mice during the administration period, which may be due to the transient weight loss in the early stage of administration caused by its side effects. Figure 7 C represents the daily weight change of mice during modeling and drug administration. The weight of mice in the blank control group, as well as the low- and high-dose Ie groups, showed an increasing trend. The weight of mice in the model group remained stable, while the weight of the positive control group showed a steady decreasing trend. In conclusion, Ie administration can significantly repair weight loss symptoms caused by depression in mice, and this effect is dose-dependent, with the repair effect increasing with increasing dose.

[0065] like Figure 8 As shown, compared with the blank control group, the model group zebrafish showed a significant increase in immobility time, immobility frequency, tortuosity, turning angle, and angular velocity. #### P<0.0001), the total distance traveled and swimming speed decreased significantly ( #### P<0.0001, indicating successful establishment of the reserpine-induced depression model in mice. Compared with the model group, all treatment groups increased spontaneous activity, total swimming distance, swimming speed, immobility time, tortuosity, turning angle, and angular velocity in mice, thus improving their depressive state. All behavioral indicators showed that the high-dose Ie group had the most significant therapeutic effect, and the low-dose Ie group was also slightly better than the positive control group.

[0066] Figure 9 A represents a cluster analysis of 50 biological samples, and B represents a cluster analysis of five experimental groups (10 biological samples in each group). The results show that compound Ie is more effective than fluoxetine in improving depressive states, and the improvement effect of the low-dose Ie group is better than that of the positive control group. The high-dose Ie group has the best efficacy, showing a dose-dependent effect.

[0067] The amount of feces an animal produces in an open area reflects its level of stress; the more feces, the higher the stress level. Figure 10 It can be seen that the number of feces in the model group mice was higher than that in the blank control group, and the difference was statistically significant. # P<0.05, indicating that the stress level of mice in the model group was higher than that of mice in the blank control group. The number of feces in all treatment groups was lower than that in the model group, especially the high-dose Ie group, which had a significantly lower number of feces than the model group. * P<0.05 indicates that it has the best improvement effect.

[0068] The number of times a mouse stands upright reflects its activity level and its ability to explore new things. Figure 11 It can be seen that, compared with the blank control group, the number of times the model group mice stood upright was significantly reduced. #### P<0.0001). Compared with the model group, mice in all treatment groups showed an increase in the number of times they stood upright in the open field test, with the high-dose Ie group showing the most significant effect. *** P<0.001), compared with the low-dose Ie group, the difference was statistically significant (P<0.001). ** P<0.01).

[0069] like Figure 12 As shown, compared with the blank control group, the model group mice had a significantly increased tail suspension immobility time. ## P<0.01). Compared with the model group, mice in all treatment groups showed a reduction in tail suspension immobility time, with the high-dose Ie group showing the most significant effect. ** P<0.01), compared with the low-dose Ie group, the difference was statistically significant (P<0.01). * (P<0.05), followed by the positive control group.

[0070] like Figure 13 As shown, compared with the blank control group, the model group mice had a significantly increased forced swimming immobility time. ### P<0.001). Compared with the model group, mice in both the low-dose and high-dose Ie groups showed a significant reduction in forced swimming immobility time. **** P<0.0001); the positive control group showed poor improvement and there was no statistically significant difference compared with the model group.

[0071] Depend on Figure 14 As shown in A, the total movement distance in the blank control group was ≥1200cm, while that in the model group was ≤900cm. Figure 14In group B, the movement speeds in the blank control group were all ≥4 cm / s, while those in the modle group were all ≤3 cm / s. If the response rate was measured by a movement distance >900 cm and a movement speed >3 cm / s, the response rate in the positive control group was 50%, in the low-dose Ie group it was 70%, and in the high-dose Ie group it was 70%. If the cure rate was measured by a movement distance ≥1200 cm and a movement speed ≥4 cm / s, the cure rate in the positive control group was 20%, in the low-dose Ie group it was 30%, and in the high-dose Ie group it was 70%. Therefore, regardless of the dose, the response rate and cure rate of compound Ie were superior to fluoxetine. Within the same administration time, compound Ie showed better antidepressant efficacy and faster onset of action.

[0072] In summary, using reserpine-induced depression models in zebrafish and mice, the antidepressant efficacy of compounds Ie and fluoxetine was evaluated using OFT, TST, and FST experiments. The results showed that both compounds Ie and fluoxetine significantly increased activity levels and the ability to explore novelties in reserpine-induced depressed mice and zebrafish models, thus improving depressive symptoms. Compound Ie demonstrated better efficacy, a higher response rate, and faster onset of action than fluoxetine, and significantly improved weight loss symptoms in the depressed mouse models. It shows promise as a novel antidepressant and warrants further investigation.

Claims

1. The use of apophyll alkaloids as shown in Formula III in the preparation of antidepressant drugs; ； 。 2. The use according to claim 1, characterized in that... Using apophene alkaloids as shown in Formula III as the active ingredient, and supplemented with a pharmaceutically acceptable carrier, any pharmaceutically acceptable dosage form can be prepared.

3. The use according to claim 2, characterized in that... The dosage form is selected from tablets, capsules, pellets, granules, powders, lozenges, aqueous or oily suspensions, and injections.

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