Use of the Rho kinase inhibitor Fasudil in suppressing hallucinations

Abstract

The present invention discloses a novel use of the Rho kinase inhibitor Fasudil in inhibiting hallucinations. The Fasudil can specifically inhibit hallucinations and has good development potential for inhibiting 5HT. 2A The prospect of drugs for the treatment of receptor-mediated hallucinations provides a theoretical basis for inhibiting the hallucinations induced by hallucinogens in the clinical treatment of mental illnesses such as depression. The clinical safety of Fasudil has been recognized, which can save the research and development costs of drugs for inhibiting hallucinations.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular, the present invention relates to a new use of the Rho kinase inhibitor Fasudil in inhibiting hallucinations. Background Art

[0002] Existing clinical antidepressants are mainly "monoamine strategy" drugs. Although they can effectively treat depression, most of them have serious defects such as delayed onset (2-6 weeks), low efficacy (50%-70%), lack of cognitive improvement or even cognitive damage, leading to sexual dysfunction and suicidal tendencies. Therefore, it is of great significance to develop new fast, efficient and low-toxic antidepressants. In recent years, classic hallucinogens have made significant progress in the treatment of mental illnesses such as depression, anxiety, and post-traumatic stress disorder, especially in promoting neuroplasticity, regulating the body's immune status, and regulating the release of neurotransmitters.

[0003] Classic hallucinogens are a class of psychoactive substances that act on 5-HT receptors, mainly through activating 5-HT 2A Receptor-induced changes in sensation and emotion, as well as hallucinations, include 2,5-dimethoxy-4-methylamphetamine (DOM), psilocybin, psilocin, 2,5-dimethoxy-4-iodoamphetamine (DOI), lysergic acid diethylamide (LSD), and N,N-dimethyltryptamine (DMT). In the "Catalogue of Psychotropic Drugs" published in 2013, classic hallucinogens such as DMT, LSD, mescaline, and psilocybin were listed as first-class psychotropic drugs. Currently, the European Medicines Agency has approved a Phase III clinical study of psilocybin for the treatment of major depressive disorder (MDD), which has shown advantages such as rapid onset and long-lasting effects in antidepressant treatment. DMT has shown good antidepressant and antianxiety effects in clinical studies. In addition, LSD also showed significant therapeutic effects on depression, drug dependence and anxiety in animal experiments. It can be seen that classic hallucinogens are very likely to replace traditional antidepressants and be widely used in the clinical treatment of depression and other mental-related diseases.

[0004] However, the side effect of hallucinations induced by classic hallucinogens has greatly limited their application in the clinical treatment of mental illnesses such as depression. Therefore, how to inhibit or reduce the occurrence of hallucinations while ensuring the therapeutic effect of classic hallucinogens is an important issue that needs to be urgently addressed in this field. Summary of the invention

[0005] In view of the technical problems existing in the above-mentioned background technology, the present invention provides a new use of the Rho kinase inhibitor Fasudil in inhibiting hallucinations. The present invention first discovered that Nogo-A / RhoA signaling pathway antagonists have significant potential to counteract the hallucinations caused by hallucinogens, and proved through animal experiments that the Rho kinase inhibitor Fasudil can specifically inhibit hallucinations.

[0006] The present invention adopts the following technical solutions to achieve the above purpose:

[0007] In a first aspect, the present invention provides use of a Rho kinase inhibitor in preparing a drug for specifically inhibiting hallucination effects.

[0008] Furthermore, the Rho kinase inhibitor is Fasudil.

[0009] Further, the hallucination effect is 5HT 2A Receptor-mediated hallucinogenic effects.

[0010] Furthermore, the drug further comprises one or more pharmaceutically acceptable inert, non-toxic excipients.

[0011] Furthermore, the excipients include carriers, solvents, emulsifiers, dispersants, wetting agents, adhesives, stabilizers, colorants, and flavors.

[0012] Furthermore, the medicine is in the form of injection, capsule, granule, drop, lyophilized product, granule or tablet.

[0013] Furthermore, the drug is prepared into injection, capsule, granule, drop, lyophilized product, granule or tablet by adding pharmaceutical auxiliary ingredients to Fasudil with a mass fraction of more than 80%.

[0014] In the present invention, the term "Rho kinase (ROCK) inhibitor" refers to a class of agents that can inhibit Rho kinase. Rho kinase is a downstream substrate of Rho protein and is one of the main kinases involved in cell movement discovered in recent years. It has an important regulatory effect on cell division, contraction, adhesion, migration, secretion and other activities. It is closely related to the occurrence and development of cardiovascular diseases such as vasospasm, arteriosclerosis, local ischemia / reperfusion injury, heart failure, myocardial infarction, hypertension and angina pectoris. Rho kinase has now become an important target for the development of new drugs related to cardiovascular diseases.

[0015] In a specific embodiment of the present invention, the Rho kinase inhibitor is Fasudil, also known as Fasudil, purchased from Shanghai Taoshu Biotechnology Co., Ltd., with the item number T3060. Fasudil is a new drug with a wide range of pharmacological effects, and its main indications are to improve and prevent cerebral vasospasm and cerebral ischemia symptoms caused after subarachnoid hemorrhage surgery. The molecular structure of Fasudil is a 5-isoquinoline sulfonamide derivative, which dilates blood vessels by increasing the activity of myosin light chain phosphatase, reduces the tension of endothelial cells, improves brain tissue microcirculation, does not produce and aggravates brain steal, and can antagonize inflammatory factors, protect nerves from apoptosis, and promote nerve regeneration. Fasudil hydrochloride has a certain effect on promoting the recovery of nerve function, alleviating clinical symptoms, and reducing disability rates.

[0016] In the present invention, the term "hallucinatory effect" refers to 5HT 2A Receptor-mediated hallucinations, i.e., classical hallucinogens act on 5-HT receptors and induce hallucinations by activating 5-HT2A receptors. The classical hallucinogens include, but are not limited to, 2,5-dimethoxy-4-methylamphetamine (DOM), psilocybin, psilocin, 2,5-dimethoxy-4-iodoamphetamine (DOI), lysergic acid diethylamide (LSD), and N,N-dimethyltryptamine (DMT). In addition, the hallucinations are not limited to the hallucinations induced by classical hallucinogens, and the pathological hallucinations induced by other hallucinogens are all within the protection scope of the classical hallucinogens described in the present invention.

[0017] In a specific embodiment of the present invention, the head shaking mouse model induced by the classic hallucinogen DOM or Psilocin is used as an example to representatively study the effect of Fasudil described in the present invention on the hallucination induced by the hallucinogen. The hallucinogen is not limited to the specific hallucinogens listed in the present invention. As long as it is a hallucinogen that can cause hallucinations, it is within the protection scope of the present invention. In a specific embodiment of the present invention, the hallucinogen is preferably a 5-HT receptor that acts on the 5-HT receptor by activating the 5-HT 2A Receptor-induced hallucinogens that produce hallucinogenic effects.

[0018] Furthermore, the head-twist mouse model used in the present invention is currently the most commonly used animal model for studying hallucinogenic behavior in the field. The head-twist response (HTR) is a rapid left-right rotation of the head. When rats and mice are given serotonergic hallucinogens or other 5-HT 2A The head-thrust response is widely used as a 5-HT 2A Behavioral assay of receptor activation. There is a strong positive correlation between the head-twitch effect of mice and the hallucinogenic effect of humans, that is, the head-twitch reaction can indicate the occurrence of hallucinogenic behavior.

[0019] Furthermore, in view of the fact that the drug described in the first aspect of the present invention can act systemically and / or locally, based on this, it can be administered in a suitable manner, such as by oral, parenteral, pulmonary or nasal routes, and the specific form of the drug described in the first aspect of the present invention can be administered in a manner suitable for these routes of administration.

[0020] The dosage form suitable for oral administration can act rapidly and / or release the drug described in the first aspect of the present invention in an improved manner, and includes crystalline and / or amorphous and / or dissolved forms, such as tablets (uncoated or coated tablets, which, for example, have a coating that resists gastric juice or delays dissolution or is insoluble, and releases according to the characteristics of the drug itself described in the first aspect of the present invention), tablets that break rapidly in the mouth, or films, films / lyophilizates, capsules (for example, hard or soft capsules), sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.

[0021] The administration form of parenteral administration can be carried out avoiding the absorption step (e.g., intravenous, intraarterial, intracardiac, intraspinal, lumbar, intraarticular) or simultaneously including absorption (e.g., intramuscular, subcutaneous, intradermal, percutaneous or intraperitoneal). Suitable administration forms suitable for parenteral administration are especially preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophilizates or sterile powders.

[0022] Suitable for another route of administration are, for example, pharmaceutical forms for inhalation, such as powder inhalation or inhalation with a nebulizer, or pharmaceutical forms which can be taken nasally, in the form of drops, solutions or sprays.

[0023] According to the first aspect of the invention, the drug described in the dosage form can be converted. This can be done in a manner known per se by mixing with inert, non-toxic, pharmacologically suitable excipients. These excipients include in particular carriers (e.g., microcrystalline cellulose, lactose, mannitol, starch), solvents (e.g., liquid polyethylene glycol), emulsifiers and dispersants or wetting agents (e.g., sodium lauryl sulfate, polyoxysorbitan oleate, propylene glycol), binders (e.g., polyvinyl pyrrolidone), synthetic and natural polymers (e.g., albumin), stabilizers (e.g., antioxidants, such as ascorbic acid), colorants (e.g., inorganic pigments, iron oxides) and masking flavors and odors.

[0024] In addition, the present invention also provides the use of Rho kinase activators in the preparation of drugs for treating analgesia, addiction, autoimmune diseases, depression and anxiety, Alzheimer's disease and related dementia, post-traumatic stress disorder, autism spectrum disorders and other mental illnesses. In a specific embodiment of the present invention, the Rho kinase activator is preferably LPA.

[0025] In a second aspect, the present invention provides a pharmaceutical composition for specifically suppressing hallucination effects.

[0026] Furthermore, the pharmaceutical composition comprises an effective amount of a Rho kinase inhibitor.

[0027] Furthermore, the Rho kinase inhibitor is Fasudil.

[0028] Furthermore, the pharmaceutical composition further comprises one or more pharmaceutically acceptable inert, non-toxic excipients.

[0029] In the present invention, the term "effective amount" refers to an amount having a therapeutic effect or an amount required to produce a therapeutic effect in a subject. For example, a drug therapeutically or pharmaceutically effective amount refers to the amount of a drug required to produce the desired therapeutic effect, and the therapeutic effect can be reflected by the results of clinical trials, model animal studies and / or in vitro studies. The pharmaceutically effective amount depends on several factors, including but not limited to the characteristic factors of the subject (such as height, weight, sex, age and medication history), the severity of the disease.

[0030] Furthermore, the pharmaceutical composition may also contain other therapeutic agents for suppressing or alleviating hallucination effects.

[0031] Furthermore, the other therapeutic agents for inhibiting or alleviating hallucinations include, but are not limited to: any currently disclosed agent that can be used to inhibit or alleviate hallucinations, and / or any currently disclosed agent that can be used to assist in inhibiting or alleviating hallucinations. The pharmaceutical composition composed of the above agents and the Rho kinase inhibitor Fasudil described in the present invention is also within the protection scope of the present invention. When the agent and the Rho kinase inhibitor Fasudil described in the present invention are used in combination, they can be used simultaneously or sequentially (first use the agent and then use the Rho kinase inhibitor Fasudil described in the present invention, or first use the Rho kinase inhibitor Fasudil described in the present invention and then use the agent). In addition, when the agent and the Rho kinase inhibitor Fasudil described in the present invention are used in combination, their administration methods can be the same or different, and the administration methods include, but are not limited to: intravenous, subcutaneous, intraperitoneal, intracranial, intrathecal, intraarterial (e.g., via the carotid artery), intramuscular, intranasal, or a combination of any of these administration methods.

[0032] Further, the administration of suitable pharmaceutical compositions includes any of various methods and delivery systems known to those skilled in the art, so that the pharmaceutical compositions of the present invention are physically introduced into the subject, including but not limited to oral administration, parenteral administration, administration by inhalation spray, topical administration, rectal administration, nasal administration, buccal administration, vaginal administration or administration by implanted drug storage device. In a specific embodiment of the present invention, administration by injection is preferred, and administration by injection includes the form of rapid injection or continuous infusion. In the case where the pharmaceutical composition is used for administration by injection, it can be in the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it can contain formulation agents, such as suspending agents, preservatives, stabilizers and / or dispersants.

[0033] In addition, the present invention also provides a method for inhibiting or alleviating hallucinations, the method comprising the following steps: administering an effective amount of the Rho kinase inhibitor Fasudil or the pharmaceutical composition according to the second aspect of the present invention to a subject in need thereof.

[0034] In a specific embodiment of the present invention, the hallucinogenic effect is preferably 5HT 2A Receptor-mediated hallucinations, that is, classic hallucinogens act on 5-HT receptors and induce hallucinations by activating 5-HT2A receptors. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The classic hallucinogens DOM and Psilocin, the non-hallucinogenic 5-HT 2A The results of the changes in the expression of Nogo-A and RhoA proteins in the cerebral cortex after intraperitoneal administration of receptor agonists Lisuride and TBG to mice;

[0036] Figure 2 The classic hallucinogens DOM and Psilocin, the non-hallucinogenic 5-HT 2A The results of the changes in phosphorylation of Nogo-A and RhoA proteins in the cerebral cortex of mice after intraperitoneal administration of receptor agonists Lisuride and TBG;

[0037] Figure 3 The results of the effect of Fasudil on the head-twitching behavior of mice induced by the classic hallucinogen DOM are shown in the figure, wherein each group has n=8-10, and the data are expressed as mean±SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, and the data were analyzed using one-way ANOVA and Dunnett's test;

[0038] Figure 4The results of the effect of Fasudil on the head-twitching behavior of mice induced by the classic hallucinogen Psilocin are shown in the figure, wherein each group has n=8-10, and the data are expressed as mean±SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, and the data were analyzed using one-way ANOVA and Dunnett's test;

[0039] Figure 5 The graph shows the effect of the P75 NTR inhibitor TAT-Pep5 on the head-twitching behavior of mice induced by the classic hallucinogen DOM, wherein n=6 in each group, data are expressed as mean±SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, and the data were analyzed using one-way ANOVA and Dunnett's test. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and are not to be construed as limiting the present invention. It will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and purposes of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The experimental methods for which specific conditions are not specified in the following examples are usually tested under conventional conditions or under conditions recommended by the manufacturer.

[0041] Example 1 Western Blot Detection of Changes in Nogo-A and RhoA after the Effect of Hallucinogens 1. Experimental Materials

[0042] The experimental reagents, experimental antibodies and experimental instruments used in this example are shown in Tables 1-3 below.

[0043] Table 1 Experimental reagents

[0044]

[0045] Table 2 Experimental antibodies

[0046]

[0047] Table 3 Experimental instruments

[0048]

[0049] The main reagent configuration in this example is:

[0050] 10× running buffer: prepare running buffer and double distilled water at a ratio of 1:9.

[0051] 10× transfer buffer: prepare transfer buffer: methanol: double distilled water in a ratio of 1:2:7.

[0052] 10×TBST: Prepare TBST and double distilled water at a ratio of 1:9.

[0053] 5% skim milk powder: Take 2 g skim milk powder and add 40 mL of 1× TBST to fully dissolve it.

[0054] 5% BSA: Take 2 g of BSA and add 40 mL of 1× TBST to fully dissolve it.

[0055] Ammonium persulfate: Take 1.00 g of ammonium persulfate and dissolve it in 10 mL of double distilled water.

[0056] 2. Western Blot detection of changes in Nogo-A and RhoA after hallucinogens

[0057] The samples used in this example are the classic hallucinogens DOM (1 mg / kg) and Psilocin (0.5 mg / kg) and the non-hallucinogenic 5-HT 2A The protein extracts of the cerebral cortex 10 minutes after intraperitoneal administration of receptor agonist Lisuride (T61065-Shanghai Taoshu Biotechnology Co., Ltd.) (0.1 mg / kg) and TBG (synthesized by the Institute of Toxicology and Pharmacology, Academy of Military Medical Sciences) (20 mg / kg) to mice (Beijing Sibeifu Biotechnology Co., Ltd., C57, male, 18-22 g, 6-8 weeks) were collected. The Western Blot experiment was used to detect the changes in the expression of Nogo-A and RhoA proteins in the above samples. The specific Western Blot experimental method is as follows:

[0058] (1) Glue making

[0059] ① Select a glass plate with a smooth bottom edge, clean the glass plate and comb, rinse them with distilled water and blow dry.

[0060] ② After aligning the thick and thin glass plates, put them into the clamp and clamp them vertically on the rack. During operation, the lower edges of the two glass plates should be aligned to avoid glue leakage.

[0061] ③ Prepare the required separation gel according to the polyacrylamide gel formula, add TEMED and shake well immediately before pouring the gel. The formula of 10% separation gel is shown in Table 4 below, and the formula of 5% concentrated gel is shown in Table 5 below.

[0062] Table 4 10% separation gel formula

[0063]

[0064]

[0065] Table 5 5% concentrated gel formula

[0066]

[0067] (2) Glue filling and sample loading

[0068] ① When pouring glue, use a gun to add glue along one side of the glass plate until the glue surface rises to about 15mm from the upper edge of the short plate. Then add a layer of water, and the glue surface will solidify faster after the liquid seal. When pouring glue, you can start faster, and slow down when the glue surface reaches the required height. The glue liquid seal should be applied slowly and evenly from left to right, otherwise the glue will be deformed.

[0069] ② When there is a broken line between the water and the glue (at room temperature, about 20 minutes), it means that the glue has solidified. Wait for another 3 minutes for the glue to fully solidify, then pour off the upper layer of water and absorb it with filter paper.

[0070] ③ Prepare 5% concentrated gel according to the polyacrylamide gel formula, add TEMED and shake well immediately to fill the gel. Fill the remaining space from one side with concentrated gel and then insert the comb into the concentrated gel. When inserting the comb, insert one side of the comb first, then slowly insert the other side, and finally check whether the comb is level.

[0071] ④After gelation, rinse the gel with distilled water and place it in the electrophoresis tank. The thin glass plate is on the inside and the thick glass plate is on the outside. If only one gel is run, a plastic plate should be placed on the other side of the electrophoresis tank to replace the glass plate.

[0072] ⑤ After filling the inner tank with new electrophoresis solution, start preparing for sample loading. The inner tank electrophoresis solution should at least cover the inner glass plate, and the outer tank should be filled with about 3 cm of electrophoresis solution to cover the bottom edge of the glass plate. Hold the two sides of the comb with both hands and gently pull it out vertically upwards. Use a 1 mL pipette to blow and rinse the sample wells. For sample loading, use a pipette to absorb the sample, insert the tip of the pipette into the gap between the two plates above the sample wells, and slowly add the sample. The wells without sample loading need to be filled with 1× loading buffer.

[0073] (3) Electrophoresis

[0074] ① Select constant voltage 80V for electrophoresis. After the sample enters the separation gel, the voltage can be adjusted to 120V to speed up the process. Electrophoresis can be terminated when bromophenol blue runs to the bottom edge of the gel.

[0075] (4) Transfer

[0076] ① Prepare a PVDF membrane with an area slightly larger than the area of ​​the rubber surface to be transferred. To transfer a piece of rubber, 8 thin filter papers or 4 thick filter papers (8×10cm) are required. If it is a PVDF membrane, it needs to be activated with methanol for 30-60s before use and the transfer solution contains methanol.

[0077] ② Place the transfer clip, two sponges, filter paper and membrane in a large glass dish with electrotransfer solution. Open the clip to keep the black side horizontal. Place a piece of sponge paper on top, press the sponge with your hand to drive out the bubbles, and let the electrotransfer solution soak the sponge. Place two layers of thick filter paper on the mat, fix the filter paper with one hand and drive out the bubbles with the other hand.

[0078] ③Pry off the glass plate and peel it off, cut off the glue deformed by squeezing at the lower edge, and peel off the concentrated glue cleanly. Carefully peel off the separation glue and cover it on the filter paper, gently remove the bubbles with your hands, and cover the membrane on the glue. After covering, it cannot be moved. Then cover it with filter paper and sponge, and clamp it with a clip.

[0079] ④ Place the clips in the electroporation tank, with the gel at the negative electrode and the membrane at the positive electrode (black-black, red-white). Use ice cubes to keep the electroporation system in a low temperature environment and transfer at a constant current of 200 mA for 2 hours.

[0080] ⑤After transfer, use tweezers to remove the membrane, rinse once with TBST, and pour from the side to prevent washing away the protein.

[0081] (5) Antibody incubation

[0082] ① First, place the membrane in blocking solution (5% skim milk or BSA) and shake it at room temperature for 1-4 hours or at 4°C overnight.

[0083] ② Dilute the primary antibody with 5% BSA at a certain ratio. Add the primary antibody diluent and shake at room temperature for 1-4 hours or at 4°C overnight.

[0084] ③ Recover the primary antibody and wash the membrane 3-4 times with TBST solution at room temperature, each time for 5 minutes.

[0085] ④ Dilute the secondary antibody with 5% milk at a certain ratio (usually 1:5000), add the secondary antibody diluent, and incubate on a shaker at room temperature for 1 hour.

[0086] ⑤Discard the secondary antibody and wash 3-4 times with TBST, 5 min each time.

[0087] (6) Development

[0088] ① Place the membrane protein side up on the blackboard, mix the developer A / B solution in a ratio of 1:1 (change the gun tip), and then drop it on the membrane to fully cover it.

[0089] ② Place the blackboard into the exposure machine, shoot the marker under white light conditions, and expose the target strip under self-luminous conditions.

[0090] (7) Statistics

[0091] Data analysis was performed using Photoshop, ImageJ, and GraphPad Prism.

[0092] 3. Experimental results

[0093] Classic hallucinogens DOM (1 mg / kg) and Psilocin (0.5 mg / kg), non-hallucinogenic 5-HT 2A The expression of Nogo-A and RhoA proteins in the protein extracts of the cerebral cortex 10 minutes after intraperitoneal administration of the receptor agonists Lisuride (0.1 mg / kg) and TBG (20 mg / kg) to mice is shown in the results. Figure 1 The results showed that the amount of Nogo-A protein did not change significantly, and the amount of RhoA protein showed an upward trend after DOM and Psilocin administration. This indicates that RhoA protein may play a role in the acute effects of classic hallucinogens by changing the amount of protein, while Nogo-A protein may not play a role by changing the amount of protein, but may play a role through protein modification or other forms of changes.

[0094] Example 2 Phos-tag gel electrophoresis to detect changes in Nogo-A and RhoA after the action of hallucinogens 1. Experimental materials

[0095] The experimental reagents, experimental antibodies and experimental instruments used in this example are shown in Tables 6-8 below.

[0096] Table 6 Experimental reagents

[0097]

[0098]

[0099] Table 7 Experimental Antibodies

[0100]

[0101] Table 8 Experimental instruments

[0102]

[0103] The main reagent configuration in this example:

[0104] 1× running buffer: prepare running buffer and double distilled water at a ratio of 1:9.

[0105] 1× transfer buffer: prepare transfer buffer, methanol and double distilled water in a ratio of 1:2:7. 1× TBST: prepare TBST and double distilled water in a ratio of 1:9.

[0106] 5% skim milk powder: Take 2 g skim milk powder and add 40 mL of 1× TBST to fully dissolve it.

[0107] 5% BSA: Take 2 g BSA and add 40 mL 1× TBST to fully dissolve it.

[0108] Ammonium persulfate: Take 1.00 g of ammonium persulfate and dissolve it in 10 mL of double distilled water.

[0109] Phosbind: 10 mg of Phosbind was dissolved in 0.10 mL of methanol and 3.2 mL of distilled water.

[0110] 10mmol / L MnCl 2+ : 10 mg dissolved in 5 mL of distilled water.

[0111] 10mmol / L EDTA: 350mg EDTA is dissolved in 120mL electrotransfer solution.

[0112] 2. Experimental methods

[0113] The samples used in this example are the classic hallucinogens DOM (1 mg / kg) and Psilocin (0.5 mg / kg) and the non-hallucinogenic 5-HT 2A Protein extracts from the cerebral cortex 10 minutes after intraperitoneal administration of receptor agonist Lisuride (T61065-Shanghai Taoshu Biotechnology Co., Ltd.) (0.1 mg / kg) and TBG (synthesized by the Institute of Toxicology and Pharmacology, Academy of Military Medical Sciences) (20 mg / kg) to mice (Beijing Sibeifu Biotechnology Co., Ltd., C57, male, 18-22 g, 6-8 weeks). Phos-tag gel electrophoresis was used to detect the phosphorylation changes of Nogo-A and RhoA proteins in the above samples. The specific Phos-tag gel electrophoresis detection experimental method is as follows:

[0114] (1) Glue making

[0115] ① Select a glass plate with a smooth bottom edge, clean the glass plate and comb, rinse them with distilled water and blow dry.

[0116] ② After aligning the thick and thin glass plates, put them into the clamp and clamp them vertically on the rack. During operation, the lower edges of the two glass plates should be aligned to avoid glue leakage.

[0117] ③ Prepare the required separation gel according to the polyacrylamide gel formula, add TEMED and shake well immediately before pouring the gel. The formula of 10% separation gel is shown in Table 9 below, and the formula of 5% concentrated gel is shown in Table 10 below.

[0118] Table 9 10% separation gel formula

[0119]

[0120] Table 10 5% concentrated gel formula

[0121]

[0122]

[0123] (2) Glue filling and sample loading

[0124] ① When pouring glue, use a gun to add glue along one side of the glass plate until the glue surface rises to about 15mm from the upper edge of the short plate. Then add a layer of water, and the glue surface will solidify faster after the liquid seal. When pouring glue, you can start faster, and slow down when the glue surface reaches the required height. The glue liquid seal should be applied slowly and evenly from left to right, otherwise the glue will be deformed.

[0125] ② When there is a broken line between the water and the glue (at room temperature, about 20 minutes), it means that the glue has solidified. Wait for another 3 minutes for the glue to fully solidify, then pour off the upper layer of water and absorb it with filter paper.

[0126] ③ Prepare 5% concentrated gel according to the polyacrylamide gel formula, add TEMED and shake well immediately to fill the gel. Fill the remaining space from one side with concentrated gel and then insert the comb into the concentrated gel. When inserting the comb, insert one side of the comb first, then slowly insert the other side, and finally check whether the comb is level.

[0127] ④After gelation, rinse the gel with distilled water and place it in the electrophoresis tank. The thin glass plate is on the inside and the thick glass plate is on the outside. If only one gel is run, a plastic plate should be placed on the other side of the electrophoresis tank to replace the glass plate.

[0128] ⑤ After filling the inner tank with new electrophoresis solution, start preparing for sample loading. The inner tank electrophoresis solution should at least cover the inner glass plate, and the outer tank should be filled with about 3 cm of electrophoresis solution to cover the bottom edge of the glass plate. Hold the two sides of the comb with both hands and gently pull it out vertically upwards. Use a 1 mL pipette to blow and rinse the sample wells. For sample loading, use a pipette to absorb the sample, insert the tip of the pipette into the gap between the two plates above the sample wells, and slowly add the sample. The wells without sample loading need to be filled with 1× loading buffer.

[0129] (3) Electrophoresis

[0130] ① Select a constant voltage of 80V for electrophoresis. After the sample enters the separation gel, the voltage can be adjusted to 120V to speed up the process. Electrophoresis can be terminated when bromophenol blue runs to the bottom edge of the gel.

[0131] (4) Membrane washing

[0132] After electrophoresis and before transfer to the membrane, a chelating agent (EDTA) is required to remove manganese ions (Mn 2+ ). This step can improve the transfer efficiency of phosphorylated and non-phosphorylated proteins to the PVDF membrane.

[0133] ① After electrophoresis, soak the gel in a common transfer buffer containing 1-10mmol / L EDTA for at least 10 minutes, while gently shaking. (10 minutes × 1-3 times). Adjust the treatment time and temperature of the EDTA buffer according to the thickness of the gel (for example: 1.5mm thick: 20 minutes × twice).

[0134] ② Soak the gel in a normal transfer buffer without EDTA for 10 minutes while gently shaking (10 minutes x 1 time).

[0135] (5) Transfer

[0136] ① Prepare a PVDF membrane with an area slightly larger than the area of ​​the rubber surface to be transferred. To transfer a piece of rubber, 8 thin filter papers or 4 thick filter papers (8×10cm) are required. If it is a PVDF membrane, it needs to be activated with methanol for 30-60s before use and the transfer solution contains methanol.

[0137] ② Place the transfer clip, two sponges, filter paper and membrane in a large glass dish with electrotransfer solution. Open the clip to keep the black side horizontal. Place a piece of sponge paper on top, press the sponge with your hand to drive out the bubbles, and let the electrotransfer solution soak the sponge. Place two layers of thick filter paper on the mat, fix the filter paper with one hand and drive out the bubbles with the other hand.

[0138] ③Pry off the glass plate and peel it off, cut off the glue deformed by squeezing at the lower edge, and peel off the concentrated glue cleanly. Carefully peel off the separation glue and cover it on the filter paper, gently remove the bubbles with your hands, and cover the membrane on the glue. After covering, it cannot be moved. Then cover it with filter paper and sponge, and clamp it with a clip.

[0139] ④ Place the clips in the electroporation tank, with the gel at the negative electrode and the membrane at the positive electrode (black-black, red-white). Use ice cubes to keep the electroporation system in a low temperature environment and transfer at a constant current of 200 mA for 2 hours.

[0140] ⑤After transfer, use tweezers to remove the membrane, rinse once with TBST, and pour from the side to prevent washing away the protein.

[0141] (6) Antibody incubation

[0142] ① First, place the membrane in blocking solution (5% skim milk or BSA) and shake it at room temperature for 1-4 hours or at 4°C overnight.

[0143] ② Dilute the primary antibody with 5% BSA at a certain ratio. Add the primary antibody diluent and shake at room temperature for 1-4 hours or at 4°C overnight.

[0144] ③ Recover the primary antibody and wash the membrane 3-4 times with TBST solution at room temperature, each time for 5 minutes.

[0145] ④ Dilute the secondary antibody with 5% milk at a certain ratio (usually 1:5000), add the secondary antibody diluent, and incubate on a shaker at room temperature for 1 hour.

[0146] ⑤Discard the secondary antibody and wash 3-4 times with TBST, 5 min each time.

[0147] (7) Development

[0148] ① Place the membrane protein side up on the blackboard, mix the developer A / B solution in a ratio of 1:1 (change the gun tip), and then drop it on the membrane to fully cover it.

[0149] ② Place the blackboard into the exposure machine, shoot the marker under white light conditions, and expose the target strip under self-luminous conditions.

[0150] (8) Statistics

[0151] Data analysis was performed using Photoshop, ImageJ, and GraphPad Prism.

[0152] 3. Experimental results

[0153] According to the Phos-Tag experimental principle, if two bands with similar distances appear for the same protein, the upper band is the amount of phosphorylated protein, and the lower band is the amount of unphosphorylated protein. The upper and lower bands are the total amount of the protein.

[0154] The phosphorylation changes of Nogo-A and RhoA proteins in the protein extract samples of the mouse cerebral cortex at 10 minutes after the classic hallucinogens DOM (1 mg / kg) and Psilocin (0.5 mg / kg), the non-hallucinogenic 5-HT2A receptor agonist Lisuride (0.1 mg / kg) and TBG (20 mg / kg) were intraperitoneally administered to mice. The results are shown in the figure. Figure 2 The results showed that only one band was detected for Nogo-A protein, and the phosphorylation change of Nogo-A protein was not obvious. Two bands were detected for RhoA protein, the upper band was the amount of phosphorylated protein, and the lower band was the amount of protein that did not undergo phosphorylation changes. It can be seen that the phosphorylation change of RhoA in the Psilocin group showed a downward trend. This shows that RhoA protein may play a role in the acute effect of classic hallucinogens by changing the protein amount and undergoing phosphorylation modification. However, Nogo-A protein may not play a role through phosphorylation modification.

[0155] Example 3 Study on the Inhibition of Hallucinations Induced by Classic Hallucinogens by Fasudil

[0156] 1. Experimental Materials

[0157] Experimental animals: SPF grade C57 mice, male, weighing 20-22 g; provided by Sibeifu (Beijing) Biotechnology Co., Ltd., experimental animal production license, SCXK (Beijing) 2019-0010. The experimental animals were housed in the Behavior Center of the Military Medical Research Institute, 8-10 per cage. The room temperature (22±2℃), humidity (40±20℃)% with 12h day and night alternation, free access to water and food, and acclimatization to the environment for three days before the experiment.

[0158] Experimental reagents: The experimental reagents used in this example are shown in Table 11 below.

[0159] Experimental equipment: The experimental equipment used in this example is shown in Table 12 below.

[0160] Table 11 Experimental reagents

[0161]

[0162] Table 12 Experimental equipment

[0163]

[0164] The main reagent configuration in this example:

[0165] DOM solution preparation: Weigh an appropriate amount of sample and dissolve it in physiological saline to prepare 0.1 mg / mL, suitable for intraperitoneal injection in mice.

[0166] Preparation of Psilocin solution: Weigh an appropriate amount of sample and dissolve it in 1% DMSO and 99% saline to prepare 0.5 mg / mL. Dilute it with saline to 0.05 mg / mL, suitable for intraperitoneal injection in mice.

[0167] Preparation of Fasudil solution, an inhibitor of Rho kinase (ROCK): Weigh an appropriate amount of sample and dissolve it in physiological saline to prepare 2 mM, and then dilute it to 1 mM and 0.2 mM for intracerebroventricular injection in mice.

[0168] Preparation of P75 NTR inhibitor TAT-Pep5 solution: Weigh an appropriate amount of sample and dissolve it in physiological saline to prepare 1 mg / mL, and then dilute it to 0.2 mg / mL and 0.04 mg / mL for intracerebroventricular injection in mice.

[0169] 2. Head-thrust reaction test verifies the inhibitory effect of Fasudil on the hallucinations induced by classical inhibitors

[0170] (1) Effect of Fasudil on hallucinations induced by classical inhibitors

[0171] The head-twist response (HTR) is a rapid side-to-side rotation of the head that occurs in rats and mice after administration of serotonergic hallucinogens or other 5-HT2A The head-thrust response is widely used as a 5-HT 2A Behavioral assay of receptor activation. In the study of classic hallucinogens, the head-twist reaction is currently the most commonly used animal model. There is a strong positive correlation between the head-twist reaction effect of mice and the hallucinogenic effect of humans, that is, the head-twist reaction can indicate the occurrence of hallucinogenic behavior.

[0172] The experimental mice described in this example were first administered intracerebroventricularly with the above-mentioned tool drug (Fasudil). After waiting for 30 minutes, the classic hallucinogen DOM or Psilocin was intraperitoneally injected. Immediately after the injection, the mice were placed in a transparent box for observation of head-shaking reaction behavior and manual counting, and the behavior test was carried out for 15 or 30 minutes.

[0173] Among them, the drug dose of DOM is 1mg / kg, which is injected by intraperitoneal injection; the dose of Psilocin is 0.5mg / kg, which is injected by intraperitoneal injection. The tool drug (Fasudil) is selected in three doses: low (1nmol / 5μL), medium (5nmol / 5μL), and high (10nmol / 5μL), all of which are injected by intracerebroventricular injection, 30 minutes before the administration of hallucinogens. The volume of intracerebroventricular injection is 5μL, and the volume of intraperitoneal injection is 0.2mL / 20g.

[0174] (2) Effects of the P75 NTR inhibitor TAT-Pep5 on the hallucinations induced by classical inhibitors

[0175] This example further verifies the effect of the P75 NTR inhibitor TAT-Pep5 on the head-shaking behavior of mice induced by the classic hallucinogen DOM. In the experiment, the drug dose of DOM was 1 mg / kg, and it was injected by intraperitoneal injection. TAT-Pep5 selected three doses of low (0.2 μg / 5 μL), medium (1 μg / 5 μL), and high (5 μg / 5 μL), all of which were injected by intracerebroventricular injection, and injected 30 minutes before DOM administration.

[0176] 3. Experimental results

[0177] The inhibitory effect of Fasudil on the hallucinations induced by classical depressants is shown in Figure 3 and Figure 4 , Figure 3 and Figure 4 The left figure in the figure shows that Fasudil has a significant inhibitory effect on head shaking behavior, and presents a dose gradient change, indicating that Fasudil can significantly inhibit the head shaking behavior of mice induced by classic hallucinogens, that is, the Fasudil of the present invention has a significant inhibitory effect on the hallucinations induced by classic hallucinogens; Figure 3 and Figure 4 The right figure shows the change in the number of head-shaking reactions within 15 or 30 minutes, with the number of head-shaking reactions accumulated every 5 minutes. The results show that the difference in the number of head-shaking reactions between the Fasudil-treated group and the control group began to be significant about 5 to 10 minutes after the administration of the hallucinogen. The above results further indicate that Fasudil can be used in the preparation of drugs that specifically inhibit hallucinations.

[0178] The results of the effect of the P75 NTR inhibitor TAT-Pep5 on the head-twitching behavior of mice induced by classic hallucinogens are shown in the figure. Figure 5 , Figure 5 The left figure shows that low, medium and high doses of TAT-Pep have no obvious inhibitory or upregulatory effects on the head shaking behavior of mice, indicating that in the Nogo-A / RhoA signaling pathway, the P75 NTR receptor and its downstream RhoA activation are not involved in the hallucination effect; Figure 5 The right picture shows the change in the number of head-shaking times within 30 minutes of the head-shaking experiment. The head-shaking times were counted every 5 minutes. It can be seen that there is no obvious difference in the head-shaking times curves of the four groups, which means that not all proteins in the Nogo-A / RhoA signaling pathway can inhibit hallucinations.

[0179] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of the Rho kinase inhibitor Fasudil in the preparation of a drug for specifically inhibiting hallucinations induced by hallucinogens, characterized in that: The hallucinogen is 2,5-dimethoxy-4-methylamphetamine or psilocybin; The hallucination is 5HT 2A Receptor-mediated hallucinogenic effects.

2. The use according to claim 1, characterized in that The medicament also contains one or more inert, non-toxic excipients that are pharmaceutically acceptable.

3. The use according to claim 2, characterized in that: The excipients include carriers, solvents, emulsifiers, dispersants, wetting agents, binders, stabilizers, colorants and / or flavors.

4. The use according to claim 3, characterized in that The medicine is in the form of injection, capsule, granule, drop, lyophilized product, granule or tablet.

5. The use according to claim 4, characterized in that The medicine is prepared into injection, capsule, granule, drop, lyophilized product, granule or tablet by adding pharmaceutical auxiliary ingredients to Fasudil with a mass fraction of more than 80%.

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