Application of andrographolide in preparation of medicine for preventing and / or treating benzodiazepine drug addiction

Abstract

The invention discloses an application of andrographolide in preparation of a medicine for preventing and / or treating benzodiazepine # imgabs0 # medicine addiction. The research finds that different doses of andrographolide can obviously prevent the formation of mouse CPP (Chlorinated Polymorphism) induced by a benzene dinitrogen # imgabs 1 # medicine namely flualprazolam and alprazolam; different doses of andrographolide can also significantly improve the withdrawal symptoms of benzene dinitrogen # imgabs2 # medicines including flualprazolam, flubrozolam and alprazolam, and do not affect the spontaneous movement ability of mice. Therefore, the andrographolide can be used for preparing the medicine for preventing and / or treating the benzodiazepine # imgabs3 # medicine addiction.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a method for preparing andrographolide for preventing and / or treating benzodiazepines. Use in drugs of drug addiction. Background Art

[0002] Andrographolide (CAS: 5508-58-7), molecular formula is C 20 H 30 O 5 , is the main active substance isolated from Andrographis paniculata, and is a diterpenoid lactone compound (Long Kaihua et al., Shaanxi Journal of Traditional Chinese Medicine, 2015, 36(3):347-348), and its structural formula is as follows (Yuan X, et al. Front Pharmacol. 2022; 13:980064):

[0003]

[0004] A large number of studies have shown that andrographolide and its analogs have multiple pharmacological effects, such as anti-tumor (Su M, et al. Drug Des Devel Ther. 2017; 11: 3333-3341), anti-depression (Geng J, et al. Toxicol Appl Pharmacol. 2019; 379: 114688), anti-viral (Gupta S, et al. Arch Virol. 2017; 162 (3): 611-623), anti-inflammatory (Yuan X, et al. Front Pharmacol. 2022; 13: 980064), antioxidant (Huang Z, et al. Cell Biol Toxicol. 2023; 39 (6): 3269-3285), and myocardial protection (Zhao Y, et al. Exp Ther Med. 2018; 3: 2138-2143), etc.

[0005] Benzodiazepines Drugs of this type include alprazolam, flualprazolam, flubrozolam, midazolam, brotizolam, haloxazolam, loprazolam, oxazolam, diazepam, quazepam, oxazepam, lorazepam, clonazepam, etc. Studies have shown that benzodiazepines These drugs share the same addictive mechanism, namely, by activating GABA containing α1 subunits in interneurons. AReceptors, relieve the inhibitory effect on dopaminergic neurons in the ventral tegmental area, and increase the firing frequency of dopaminergic neurons in the ventral tegmental area (Tan KR, et al. Nature. 2010; 463(7282):769-74). Long-term use of such drugs will lead to tolerance and dependence. Discontinuation of the drug will cause adverse reactions such as insomnia and anxiety (Chen Zhong, Du Junrong, et al., Pharmacology 9th Edition, 2022; 110).

[0006] In previous studies, andrographolide has mainly focused on anti-tumor, anti-inflammatory, antiviral, antioxidant, and myocardial protection. It has not yet been used to prevent and / or treat benzodiazepines. The addictive effects of drugs. Summary of the invention

[0007] Purpose of the invention: The purpose of the present invention is to provide a method for preparing andrographolide for preventing and / or treating benzodiazepines. Use in drugs of drug addiction.

[0008] The present invention found that different doses of andrographolide can significantly prevent the The formation of CPP in mice induced by fluxazolam and alprazolam in the same class of drugs was inhibited, indicating that it has the potential to prevent the addiction of fluxazolam and alprazolam. Different doses of andrographolide can also significantly improve the The results showed that andrographolide can reduce the withdrawal symptoms of fluxaprazolam, flubrozolam and alprazolam, and does not affect the spontaneous movement ability of mice, indicating that it has the potential to treat the withdrawal of fluxaprazolam, flubrozolam and alprazolam. Therefore, andrographolide can be used to prepare drugs for the prevention and / or treatment of benzodiazepines. Drugs that cause addiction.

[0009] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0010] The invention provides a method for preparing andrographolide for preventing benzodiazepines Use in drugs of drug addiction.

[0011] The benzodiazepine This type of drug includes fluphenazine and alprazolam.

[0012] The andrographolide can be used as the sole active ingredient to prepare the prevention of benzodiazepine Drugs for drug addiction; can also be used in combination with other active ingredients to prepare drugs to prevent benzodiazepines Drugs that cause addiction.

[0013] The andrographolide blocks the benzodiazepine The formation of CPP in mice induced by drugs.

[0014] The present invention also provides a method for preparing andrographolide for treating benzodiazepines. Use in drugs of drug addiction.

[0015] The benzodiazepine This type of drug includes fluxazolam, flubrozolam, and alprazolam.

[0016] The invention discloses a method for treating benzodiazepines by using andrographolide as the only active ingredient. Drugs for drug addiction; can also be used in combination with other active ingredients to prepare drugs for the treatment of benzodiazepines Drugs that cause addiction.

[0017] Andrographolide significantly improves benzodiazepine The drug-induced withdrawal symptoms were not affected and the spontaneous movement ability of mice was not affected.

[0018] The medicine comprises andrographolide and pharmaceutically acceptable excipients.

[0019] The auxiliary materials include one or more of emulsifiers, antioxidants, wetting agents, preservatives, diluents, disintegrants or adhesives.

[0020] The emulsifier is selected from at least one of Tweens, Spans, glycerol fatty acid esters, pectin, agar, sodium alginate or silicon dioxide.

[0021] The antioxidant is selected from at least one of ascorbic acid, sulfite, bisulfite, gallic acid and lipids thereof.

[0022] The wetting agent is selected from at least one of water and ethanol.

[0023] The preservative is selected from at least one of benzoic acid and its salts, sorbic acid and its salts or parabens.

[0024] The diluent is selected from at least one of starch, sugar, cellulose or inorganic salts.

[0025] The disintegrant is selected from at least one of starch, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose or cross-linked polyvinyl pyrrolidone.

[0026] The binder is selected from at least one of starch slurry, sodium carboxymethyl cellulose, povidone, hydroxypropyl cellulose, methyl cellulose or ethyl cellulose.

[0027] The dosage form of the drug is capsule, tablet, granule, oral solution, injection or infusion, etc.

[0028] The effective dosage of andrographolide is 10-90 mg / kg.

[0029] The drug of the present invention can be administered in various known ways, such as oral administration or injection. The drug of the present invention can be administered alone or in combination with other drugs. The oral composition can be any oral acceptable dosage form, including but not limited to capsules, tablets, granules, pills, suspensions and solutions.

[0030] Sterile injectable compositions can be formulated according to techniques known in the art using suitable dispersants or wetting agents and suspending agents. Pharmaceutically acceptable carriers and solvents that can be used include water, sodium chloride solution, and the like.

[0031] The actual dosage level of the active ingredient in the medicament of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration, and that is non-toxic to the patient. The selected dosage level depends on a variety of factors, including the route of administration, time of administration, rate of excretion, duration of treatment, other drugs, compounds and / or materials used in combination with andrographolide, age, sex, weight, general health and past medical history of the patient being treated, and similar factors well known in the medical field.

[0032] The present invention utilizes intraperitoneal injection of flualapzolam (4 mg / kg) and flubrozolam solution (3 mg / kg) for 28 consecutive days, and then the mice are placed in a breeding cage for normal breeding and natural withdrawal for 7 days to establish a flualapzolam and flubrozolam withdrawal mouse model, and different doses of andrographolide are administered by gavage during the withdrawal period for intervention, and the effect of andrographolide on the withdrawal symptoms of mice is evaluated through open field test (OFT) and elevated plus maze (EPM) experiments.

[0033] The present invention uses intraperitoneal injection of alprazolam solution (10 mg / kg) for 21 consecutive days, and then places the mice in a breeding cage for normal feeding and natural withdrawal for 7 days to establish an alprazolam withdrawal mouse model. During the withdrawal period, different doses of andrographolide are administered by gavage for intervention, and the effect of andrographolide on the withdrawal symptoms of mice is evaluated through the Open Field Test (OFT) and the Elevated Plus Maze (EPM) experiment.

[0034] The present invention uses the Conditioned Place Preference (CPP) experiment combined with the intraperitoneal injection of a fluprazolam solution (4 mg / kg) to establish a fluprazolam addiction mouse model, and simultaneously administers different doses of andrographolide by oral gavage for intervention, and the addiction of the mice in the model group and the drug-treated group is evaluated by the CPP experiment.

[0035] The invention uses CPP experiment combined with intraperitoneal injection of alprazolam solution (10 mg / kg) to establish an alprazolam addiction mouse model, and simultaneously intragastric administration of different doses of andrographolide is performed for intervention, and the addiction of the model group and the drug-treated group mice is evaluated through the CPP experiment.

[0036] Beneficial effects:

[0037] The andrographolide of the present invention can be used as an active ingredient for preventing and / or treating benzodiazepines. Addiction to benzodiazepines, especially fluxazolam, flubrozolam and alprazolam, has opened up new uses for andrographolide for the prevention and / or treatment of benzodiazepines. It provides new options for treating drug addiction.

[0038] The compound andrographolide of the present invention is administered orally to prevent the benzodiazepine The formation of CPP in mice induced by benzodiazepines can be prevented At the same time, it significantly improves benzodiazepine Therefore, the andrographolide of the present invention can be used to prepare a drug for preventing and / or treating benzodiazepines. Drugs that cause addiction. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0040] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0041] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0042] Andrographolide, content ≥98%, batch number: K2125557, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and prepared to the required concentration with 0.5% CMC-Na solution before use.

[0043] Flualaprozolam (CAS No.: 28910-91-0) is provided by the Joint Laboratory of Key Drug Control Technologies of the Office of the National Narcotics Control Commission and China Pharmaceutical University, with a content of ≥98%.

[0044] Flualprazolam solution: 1.6 mg of flualprazolam was dissolved in 40 μl of DMSO to prepare a stock solution, 160 μl of 20% hydroxypropyl-β-cyclodextrin (HPCD) solution was added, and the solution was diluted to 4 ml with sterile saline. The dosage volume was 0.1 ml / 10 g.

[0045] Flubrozolam (CAS No.: 612526-40-6) is provided by the Joint Laboratory of Key Drug Control Technologies of the Office of the National Narcotics Control Commission and China Pharmaceutical University, with a content of ≥98%.

[0046] Flubrozolam solution: 1.2 mg of flubrozolam was dissolved in 30 μl of DMSO to prepare a stock solution, 120 μl of 20% HPCD solution was added, and the solution was diluted to 4 ml with sterile saline. The dosage volume was 0.1 ml / 10 g.

[0047] Alprazolam (CAS No.: 28981-97-7) is provided by the Joint Laboratory of Key Drug Control Technologies of the Office of the National Narcotics Control Commission and China Pharmaceutical University, with a content of ≥98%.

[0048] Alprazolam solution: 4 mg of alprazolam is dissolved in 80 μl of DMSO to prepare a stock solution, which is then diluted to 4 ml with sterile saline and used immediately. The dosing volume is 0.1 ml / 10 g.

[0049] 0.9% Sodium Chloride Injection, batch number: 2405228, produced by Jiangsu Huaian Shuanghe Pharmaceutical Co., Ltd.

[0050] Example 1 Effect of andrographolide on fluxazolam withdrawal symptoms

[0051] Experimental animals: Male C57BL / 6J mice, weighing 20-24 g, with production license number SCXK(Su)2024-0001, and quality inspection provided by Suzhou Soochow Health and Environmental Technology Research Co., Ltd.

[0052] The laboratory room temperature is 20-22℃, the relative humidity is 40%-60%, the light is 12h / day, the cages are kept, 6 animals per cage, and the cages are cleaned every three days.

[0053] Experimental methods:

[0054] The mice were randomly divided into five groups according to their body weight, namely, normal control group (n=10), negative control group (n=9), andrographolide 10 mg / kg group (n=10), andrographolide 30 mg / kg group (n=10), andrographolide 90 mg / kg group (n=10).

[0055] The mice in the normal control group were intraperitoneally injected with an equal amount of control solution (40 μl DMSO + 160 μl 20% HPCD solution, diluted to 4 ml with sterile saline) every day, and the mice in the other groups were intraperitoneally injected with fluxazolam solution (4 mg / kg) every day, once a day, for 28 consecutive days. From the 29th day of the experiment, the intraperitoneal injection of fluxazolam solution was stopped, and the mice in the normal control group and the negative control group were gavaged with an equal amount of solvent (0.5% CMC-Na solution) every day, and the mice in the andrographolide group were gavaged with andrographolide 10, 30, 90 mg / kg every day according to the grouping situation, once a day, for 7 consecutive days.

[0056] Then the open field test (OFT) and elevated plus maze test (EPM) were carried out.

[0057] In OFT, mice were transferred to the test room in advance to adapt for more than 1 hour. The test apparatus consisted of a blue plastic box (42×42×40 cm 3 ) and the Jiliang DigBehv animal behavior analysis system for data collection. The mouse was gently placed in the center of the box and moved freely. The movement trajectory within 5 minutes was recorded by the video analysis and acquisition system. The center residence time and total movement distance of the mouse in the last 4 minutes were analyzed. The environment was kept quiet during the test. After each mouse test, the excrement was cleaned in time and the inside of the box was wiped with 75% ethanol.

[0058] In the EPM experiment, the test device consists of two open arms (30cm×5cm) and two closed arms (30cm×5cm×15cm), and is 40-55cm above the ground. The data is collected by the Jiliang DigBehv animal behavior analysis system. At the beginning of the experiment, the mouse is placed in the maze from the central grid to the open arm, and the movement trajectory within 5 minutes is recorded. The proportion of time that the experimental mouse stays in the open arm after 4 minutes is analyzed. The proportion of open arm stay time (%) = total open arm stay time / total experimental time. Keep the environment quiet during the test. After the test of each mouse, clean up the excrement in time and wipe it with 75% ethanol.

[0059] The experimental data were processed by Graph Pad Prism 9.0.0. The experimental data results are shown in 1, 2, and 3.

[0060] Table 1 Effects of andrographolide on OFT in fluxazolam withdrawal model mice

[0061]

[0062] *P<0.05, **P<0.01, ***P<0.001, compared with the negative control group

[0063] Table 2 Effects of andrographolide on OFT in fluxazolam withdrawal model mice

[0064]

[0065] # P>0.05, compared with the negative control group

[0066] Table 3 Effects of andrographolide on EPM experiment of fluxazolam withdrawal model mice

[0067]

[0068] *P<0.05, ***P<0.001, compared with the negative control group

[0069] The experimental results showed that in OFT, compared with the negative control group, the mice spent significantly more time in the center after intragastric administration of 10, 30, and 90 mg / kg andrographolide (P < 0.05, P < 0.01, and P < 0.001), but had no effect on the total distance of movement of the mice (P > 0.05), as shown in Tables 1 and 2. In the EPM experiment, compared with the negative control group, the mice spent significantly more time in the open arms after intragastric administration of 10, 30, and 90 mg / kg andrographolide (P < 0.05, P < 0.001, and P < 0.001), as shown in Table 3. The above experimental results show that andrographolide can significantly improve the anxiety-like behavior of fluxazolam withdrawal mice, and does not affect the spontaneous activity ability of mice.

[0070] Example 2 Effect of andrographolide on flubrozolam withdrawal symptoms

[0071] Experimental animals: Male C57BL / 6J mice, weighing 20-24 g, with production license number SCXK(Su)2024-0001, and quality inspection provided by Suzhou Soochow Health and Environmental Technology Research Co., Ltd.

[0072] The laboratory room temperature is 20-22℃, the relative humidity is 40%-60%, the light is 12h / day, the cages are kept, 6 animals per cage, and the cages are cleaned every three days.

[0073] Experimental methods:

[0074] The mice were randomly divided into five groups according to their body weight, namely, normal control group (n=10), negative control group (n=9), andrographolide 10 mg / kg group (n=9), andrographolide 30 mg / kg group (n=10), andrographolide 90 mg / kg group (n=9).

[0075] The mice in the normal control group were intraperitoneally injected with an equal amount of control solution (30 μl DMSO + 120 μl 20% HPCD solution, diluted to 4 ml with sterile saline) every day, and the mice in the other groups were intraperitoneally injected with flubrozolam solution (3 mg / kg) every day, once a day, for 28 consecutive days. From the 29th day of the experiment, the intraperitoneal injection of flubrozolam solution was stopped, and the mice in the normal control group and the negative control group were gavaged with an equal amount of solvent (0.5% CMC-Na solution) every day, and the mice in the andrographolide group were gavaged with andrographolide 10, 30, 90 mg / kg every day according to the grouping situation, once a day, for 7 consecutive days.

[0076] Then, open field test (OFT) and elevated plus maze test (EPM) were performed. The experimental method was the same as that in Example 1.

[0077] In OFT, the video analysis and acquisition system was used to record the movement trajectory of mice within 5 minutes, and the central residence time and total movement distance of mice in the last 4 minutes were obtained through system analysis. In the EPM experiment, the video analysis and acquisition system was used to record the movement trajectory of mice within 5 minutes, and the proportion of time that mice stayed in the open arms in the last 4 minutes was obtained through system analysis. The data of this experiment were processed by Graph Pad Prism 9.0.0. The experimental data results are shown in Tables 4, 5, and 6.

[0078] Table 4 Effects of andrographolide on OFT in flubrozolam withdrawal model mice

[0079]

[0080] *P<0.05, ***P<0.001, compared with the negative control group

[0081] Table 5 Effect of andrographolide on OFT in flubrozolam withdrawal model mice

[0082]

[0083] # P>0.05, compared with the negative control group

[0084] Table 6 Effects of andrographolide on EPM experiment of flubrozolam withdrawal model mice

[0085]

[0086] *P<0.05, **P<0.01, ***P<0.001, compared with the negative control group

[0087] The experimental results showed that in OFT, compared with the negative control group, the mice spent significantly more time in the center after intragastric administration of 10, 30, and 90 mg / kg andrographolide (P < 0.05, P < 0.05, P < 0.001), but had no effect on the total distance of movement of the mice (P > 0.05), see Tables 4 and 5. In the EPM experiment, compared with the negative control group, the mice spent significantly more time in the open arms after intragastric administration of 10, 30, and 90 mg / kg andrographolide (P < 0.01, P < 0.05, P < 0.001), see Table 6. The above experimental results show that andrographolide can significantly improve the anxiety-like behavior of flubrozolam withdrawal mice, and does not affect the spontaneous activity ability of mice.

[0088] Example 3 Effect of andrographolide on alprazolam withdrawal symptoms

[0089] Experimental animals: Male C57BL / 6J mice, weighing 20-24 g, with production license number SCXK(Su)2024-0001, and quality inspection provided by Suzhou Soochow Health and Environmental Technology Research Co., Ltd.

[0090] The laboratory room temperature is 20-22℃, the relative humidity is 40%-60%, the light is 12h / day, the cages are kept, 6 animals per cage, and the cages are cleaned every three days.

[0091] Experimental methods:

[0092] The mice were randomly divided into five groups according to their body weight, namely, normal control group (n=9), negative control group (n=9), andrographolide 10 mg / kg group (n=9), andrographolide 30 mg / kg group (n=10), andrographolide 90 mg / kg group (n=9).

[0093] The mice in the normal control group were intraperitoneally injected with an equal amount of control solution (80 μl DMSO diluted to 4 ml with sterile saline) every day, and the mice in the other groups were intraperitoneally injected with alprazolam solution (10 mg / kg) every day, once a day, for 21 consecutive days. Starting from the 22nd day of the experiment, the intraperitoneal injection of alprazolam solution was stopped, and the mice in the normal control group and the negative control group were gavaged with an equal amount of solvent (0.5% CMC-Na solution) every day, and the mice in the andrographolide group were gavaged with andrographolide 10, 30, and 90 mg / kg every day according to the grouping situation, once a day, for 7 consecutive days.

[0094] Then, open field test (OFT) and elevated plus maze test (EPM) were performed. The experimental method was the same as that in Example 1.

[0095] In OFT, the video analysis and acquisition system was used to record the movement trajectory of mice within 5 minutes, and the central residence time and total movement distance of mice in the last 4 minutes were obtained through system analysis. In the EPM experiment, the video analysis and acquisition system was used to record the movement trajectory of mice within 5 minutes, and the proportion of time that mice stayed in the open arms in the last 4 minutes was obtained through system analysis. The data of this experiment were processed by Graph Pad Prism 9.0.0. The experimental data results are shown in Tables 7, 8, and 9.

[0096] Table 7 Effects of andrographolide on OFT in alprazolam withdrawal model mice

[0097]

[0098] **P<0.01, ***P<0.001, compared with the negative control group

[0099] Table 8 Effect of andrographolide on OFT in alprazolam withdrawal model mice

[0100]

[0101] # P>0.05, compared with the negative control group

[0102] Table 9 Effects of andrographolide on EPM experiment in alprazolam withdrawal model mice

[0103]

[0104]

[0105] *P<0.05, ***P<0.001, compared with the negative control group

[0106] The experimental results showed that in OFT, compared with the negative control group, the mice spent significantly more time in the center after intragastric administration of 10, 30, and 90 mg / kg andrographolide (P < 0.01, P < 0.01, P < 0.001), but had no effect on the total distance of movement of the mice (P > 0.05), as shown in Tables 7 and 8. In the EPM experiment, compared with the negative control group, the mice spent significantly more time in the open arms after intragastric administration of 10, 30, and 90 mg / kg andrographolide (P < 0.05, P < 0.001, P < 0.001), as shown in Table 9. The above experimental results show that different doses of andrographolide can significantly improve the anxiety-like behavior of alprazolam withdrawal mice, and do not affect the spontaneous activity ability of mice.

[0107] Example 4 Effect of andrographolide on fluphenazolane-induced addiction behavior in mice

[0108] Experimental animals: Male C57BL / 6J mice, weighing 20-24 g, with production license number SCXK(Su)2024-0001, and quality inspection provided by Suzhou Soochow Health and Environmental Technology Research Co., Ltd.

[0109] The laboratory room temperature is 20-22℃, the relative humidity is 40%-60%, the light is 12h / day, the cages are kept, 6 animals per cage, and the cages are cleaned every three days.

[0110] Experimental methods:

[0111] First, the experimental mice were tested for baseline. The gates between the boxes were opened and the mice were placed in the middle box. ANY-maze software was used for data collection to record the shuttle paths of the mice within 15 minutes of free activity and the time they stayed in each box. Mice with a natural preference for the gray box were screened out based on the CPP score.

[0112] CPP score = T (striped room stay time) - T (gray room stay time); CPP score < 0, gray room preferred. Therefore, the striped room was set as a drug-accompanying box, and the gray room was set as a non-drug-accompanying box.

[0113] Subsequently, the experimental mice that naturally preferred the gray chamber were randomly divided into 5 groups: normal control group (n=9), negative control group (n=9), andrographolide 10 mg / kg group (n=9), andrographolide 30 mg / kg group (n=10), andrographolide 90 mg / kg group (n=10).

[0114] One hour after the experimental mice were gavaged with andrographolide (10, 30, 90 mg / kg), fluxazolam solution (4 mg / kg) was injected intraperitoneally, and the experimental mice were placed in the corresponding box for training and learning for 40 minutes (the gate was closed to avoid shuttling), once a day for 8 days, and the dosing schedule is detailed in Table 10. The test was carried out on the first day after the training. The mice were placed in the middle box and allowed to shuttle freely for 15 minutes. The behavioral analysis software recorded the movement trajectory and the time spent in each box. According to the time spent in the boxes on both sides, the CPP score was calculated and statistically analyzed. The experimental data were all processed with Graph Pad Prism 9.0.0. The experimental data results are shown in Table 11.

[0115] Table 10 Dosing schedule for the experiment of andrographolide intervening in the CPP of fluxazolam addiction model mice

[0116]

[0117] Note: Vehicle indicates control solution: 40 μl DMSO + 160 μl 20% HPCD solution, diluted to 4 ml with sterile saline; CMC-Na indicates 0.5% CMC-Na solution.

[0118] Table 11 Effects of andrographolide on CPP scores of alprazolam addiction model mice

[0119]

[0120] **P<0.01, ***P<0.001, compared with the negative control group

[0121] The experimental results showed that in the CPP experiment, compared with the negative control group, the CPP scores of mice were significantly decreased after intragastric administration of 10, 30, and 90 mg / kg andrographolide during the training period (P < 0.01, P < 0.01, P < 0.001), as shown in Table 11. The experimental results showed that andrographolide can prevent mice from becoming addicted to fluxazolam.

[0122] Example 5 Effect of andrographolide on alprazolam-induced addiction behavior in mice

[0123] Experimental animals: Male C57BL / 6J mice, weighing 20-24 g, with production license number SCXK(Su)2024-0001, and quality inspection provided by Suzhou Soochow Health and Environmental Technology Research Co., Ltd.

[0124] The laboratory room temperature is 20-22℃, the relative humidity is 40%-60%, the light is 12h / day, the cages are kept, 6 animals per cage, and the cages are cleaned every three days.

[0125] Experimental methods:

[0126] First, the experimental mice were tested for baseline. The gates between the boxes were opened and the mice were placed in the middle box. ANY-maze software was used for data acquisition to record the shuttle paths of the mice within 15 minutes of free activity and the time they stayed in each box. Mice with a natural preference for the gray box were screened out based on the CPP score. CPP score = T (striped room stay time) - T (gray room stay time); CPP score < 0, preference for the gray room. Therefore, the striped room was set as a drug-accompanying box, and the gray room was set as a non-drug-accompanying box. Subsequently, the experimental mice with a natural preference for the gray room were randomly divided into 5 groups: normal control group (n = 10), negative control group (n = 9), andrographolide 10 mg / kg group (n = 9), andrographolide 30 mg / kg group (n = 10), andrographolide 90 mg / kg group (n = 9).

[0127] One hour after the experimental mice were gavaged with andrographolide (10, 30, 90 mg / kg), alprazolam solution (10 mg / kg) was intraperitoneally injected, and the experimental mice were placed in the corresponding box for training and learning for 40 minutes (the gate was closed to avoid shuttling), 1 time / day training, for 8 days, the dosing schedule is detailed in Table 12. After the training, the test was carried out on the first day. The mice were placed in the middle box and allowed to shuttle freely for 15 minutes. The behavioral analysis software recorded the movement trajectory and the time spent in each box. According to the time spent in the boxes on both sides, the CPP score was calculated and statistically analyzed. The data of this experiment were all processed with Graph Pad Prism9.0.0. The experimental data results are shown in Table 13.

[0128] Table 12 Dosing schedule for the experiment of andrographolide intervening in CPP of alprazolam addiction model mice

[0129]

[0130]

[0131] Table 13 Effects of andrographolide on CPP scores of alprazolam addiction model mice

[0132]

[0133] *P<0.05, **P<0.01, ***P<0.001, compared with the negative control group

[0134] The experimental results showed that in the CPP experiment, compared with the negative control group, the CPP scores of mice were significantly decreased after intragastric administration of 10, 30, and 90 mg / kg andrographolide during the training period (P < 0.05, P < 0.01, P < 0.01), as shown in Table 13. The experimental results showed that andrographolide can prevent mice from becoming addicted to alprazolam.

[0135] In summary, in the flubrozolam natural withdrawal model mice, compared with the negative control group, after administration of 10, 30, and 90 mg / kg andrographolide, the mice's open field center movement time in OFT was significantly increased (P<0.05, P<0.01, P<0.001), there was no significant difference in the movement distance of mice in each group (P>0.05), and the proportion of time spent in the open arm in the EPM experiment was significantly increased (P<0.05, P<0.001, P<0.001); in the flubrozolam natural withdrawal model mice, compared with the negative control group, after administration of 10, 30, and 90 mg / kg andrographolide, the mice's open field center movement time in OFT was significantly increased (P<0.05, P<0.001, P<0.001). 0.05, P<0.001), there was no significant difference in the movement distance of mice in each group (P>0.05), and the proportion of time spent in the open arms in the EPM experiment was significantly increased (P<0.01, P<0.05, P<0.001); in the alprazolam natural withdrawal model mice, compared with the negative control group, after administration of 10, 30, and 90 mg / kg andrographolide, the mice's movement time in the center of the open field in OFT was significantly increased (P<0.01, P<0.01, P<0.001), there was no significant difference in the movement distance of mice in each group (P>0.05), and the proportion of time spent in the open arms in the EPM experiment was significantly increased (P<0.05, P<0.001, P<0.001). In the alprazolam addiction model mice, the preference scores of mice in CPP were significantly reduced after administration of 10, 30, and 90 mg / kg andrographolide compared with the negative control group (P<0.01, P<0.01, P<0.001). In the alprazolam addiction model mice, the preference scores of mice in CPP were significantly reduced after administration of 10, 30, and 90 mg / kg andrographolide compared with the negative control group (P<0.05, P<0.01, P<0.01).

[0136] These results suggest that andrographolide can be used in the preparation of drugs for the prevention and / or treatment of benzodiazepines Drugs that cause addiction.

[0137] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. Andrographolide in the preparation of benzodiazepines Use in drugs of drug addiction.

2. The use according to claim 1, characterized in that The benzodiazepine This type of drug includes fluphenazine and alprazolam.

3. The use according to claim 1, characterized in that The invention discloses a method for preventing benzodiazepines by using andrographolide as the only active ingredient. Drugs that cause addiction.

4. The use according to claim 1, characterized in that The andrographolide blocks the benzodiazepine The formation of CPP in mice induced by drugs.

5. Andrographolide in the preparation of benzodiazepines Use in drugs of drug addiction.

6. The use according to claim 5, characterized in that The benzodiazepine This type of drug includes fluxazolam, flubrozolam, and alprazolam.

7. The use according to claim 5, characterized in that The invention discloses a method for treating benzodiazepines by using andrographolide as the only active ingredient. Drugs that cause addiction.

8. The use according to claim 5, characterized in that The andrographolide significantly improved the The drug-induced withdrawal symptoms were not affected and the spontaneous movement ability of mice was not affected.

9. The use according to any one of claims 1 to 8, characterized in that The medicine comprises andrographolide and pharmaceutically acceptable excipients.

10. The use according to any one of claims 1 to 8, characterized in that The effective dosage of andrographolide is 10-90 mg / kg.

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