CN117618442A - A new use of the Rho kinase inhibitor Fasudil in suppressing hallucinations - Google Patents

Abstract

The present invention discloses a new use of Rho kinase inhibitor Fasudil in inhibiting hallucinations. Fasudil can specifically inhibit hallucinations and has good prospects of being developed into a drug that inhibits hallucinations mediated by 5HT _2A receptors. It is It provides a theoretical basis for inhibiting the hallucinations induced by hallucinogens in the clinical treatment of depression and other mental-related diseases, and Fasudil's clinical safety has been recognized, which can save R&D costs in the development of drugs that inhibit hallucinations.

Description

A new use of the Rho kinase inhibitor Fasudil in suppressing hallucinations

Technical field

The present invention belongs to the field of biomedical technology. Specifically, the present invention relates to the new use of Rho kinase inhibitor Fasudil in inhibiting hallucinations.

Background technique

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

Classical hallucinogens are a class of psychoactive substances that act on 5-HT receptors, inducing sensory and emotional changes and hallucinatory effects mainly by activating 5-HT _2A receptors, including 2,5-dimethoxy-4- Methamphetamine (DOM), psilocybin (Psilocybin), psilocin (Psilocin), 2,5-dimethoxy-4-iodoamphetamine (DOI), lysergic acid diethylamide (LSD), N, N-dimethyltryptamine (DMT), China’s 2013 “Catalogue of Psychotropic Drugs” lists classic hallucinogens such as DMT, LSD, mescaline and psilocybin as the first category of psychotropic drugs. At present, the European Medicines Agency has approved the Phase III clinical study of psilocibine in the treatment of major depressive disorder (MDD). It has the advantages of rapid onset and long-lasting effect in antidepressant treatment. DMT has shown good antidepressant and antianxiety effects in clinical studies. In addition, LSD has also shown significant therapeutic effects on depression, drug dependence and anxiety in animal experimental studies. It can be seen that classic hallucinogens are very promising to replace traditional antidepressants in the clinical treatment of depression and other mental-related diseases. Be widely used.

However, the side effect of hallucinations induced by classic hallucinogens greatly limits their application in the clinical treatment of depression and other mental-related diseases. Therefore, how can we suppress or suppress the effects of classic hallucinogens while ensuring their therapeutic effects? Reducing the occurrence of hallucinations is an important issue that needs to be solved urgently in this field.

Contents of the invention

In view of the technical problems existing in the above background art, the present invention provides a new use of Rho kinase inhibitor Fasudil in inhibiting hallucinations. The present invention discovered for the first time that Nogo-A/RhoA signaling pathway antagonists have significant potential to combat hallucinations caused by hallucinogens, and animal experiments have proven that the Rho kinase inhibitor Fasudil can specifically inhibit hallucinations.

The present invention adopts the following technical solutions to achieve the above objectives:

In a first aspect, the present invention provides the use of Rho kinase inhibitors in the preparation of drugs that specifically inhibit hallucinations.

Further, the Rho kinase inhibitor is Fasudil.

Further, the hallucination effect is a hallucination effect mediated by 5HT _2A receptors.

Furthermore, the medicine also contains one or more pharmaceutically acceptable inert, non-toxic excipients.

Further, the excipients include carriers, solvents, emulsifiers, dispersants, wetting agents, adhesives, stabilizers, colorants, and flavors.

Further, the medicine is an injection, capsule, granule, drop, lyophilizate, granule or tablet.

Furthermore, the medicine is an injection, capsule, granule, drop, lyophilizate, granule or tablet prepared by adding pharmaceutical auxiliary ingredients to Fasudil with a mass fraction of more than 80%.

In the present invention, the term "Rho kinase (ROCK) inhibitor" refers to a class of agents capable of inhibiting Rho kinase. Rho kinase is a downstream substrate of Rho protein. It is one of the major kinases discovered in recent years to participate in cell movement. It plays an important regulatory role in 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, ischemia/reperfusion injury, heart failure, myocardial infarction, hypertension and angina. Rho kinase has now become an important factor in the research and development of new drugs related to cardiovascular diseases. target.

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 product number of T3060. Fasudil is an inhibitor with a wide range of pharmacological effects. The main indication of this new drug is 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. By increasing the activity of myosin light chain phosphatase, it expands blood vessels, reduces the tension of endothelial cells, improves brain tissue microcirculation, and does not produce or aggravate brain blood theft. At the same time, it can antagonize inflammatory factors, protect nerves from apoptosis, and promote nerve regeneration. Fasudil hydrochloride has certain effects on promoting the recovery of nerve function, alleviating clinical symptoms, and reducing the disability rate.

In the present invention, the term "hallucinatory effect" refers to the hallucinatory effect mediated by 5HT _2A receptors, that is, the hallucination effect induced by classic hallucinogens acting on 5-HT receptors and activating 5-HT2A receptors. The classic hallucinogens include, but are not limited to: 2,5-dimethoxy-4-methamphetamine (DOM), psilocybin, psilocin, 2,5-dimethylamphetamine Oxy-4-iodoamphetamine (DOI), lysergic acid diethylamide (LSD), N,N-dimethyltryptamine (DMT). In addition, the hallucination effect is not limited to the hallucination effect induced by classic hallucinogens. The pathological hallucination effects induced by other hallucinogens are within the protection scope of the classic hallucinogens described in the present invention.

In the specific embodiment of the present invention, the present invention takes the head shaking mouse model induced by the classic hallucinogen DOM or Psilocin as an example to representatively study the effect of Fasudil of the present invention on the hallucination effect induced by hallucinogens. , the hallucinogens are not limited to the specific hallucinogens listed in the present invention, as long as they are hallucinogens that can cause hallucinations, they are within the protection scope of the present invention. In a specific embodiment of the invention, the hallucinogen is preferably a classic hallucinogen that acts on 5-HT receptors to induce hallucinogenic effects by activating 5-HT _2A receptors.

Furthermore, the head-shaking mouse model used in the present invention is currently the most commonly used animal model in this field for studying hallucinogenic behavior. The head toss response (HTR) is a rapid side-to-side rotation of the head that occurs after administration of serotonergic hallucinogens or other 5-HT _2A agonists to rats and mice. The head toss response is widely used as a 5 - Behavioral assay of HT _2A receptor activation. There is a strong positive correlation between the head shaking reaction effect in mice and the hallucinogenic effect in humans, that is, the head shaking reaction can indicate the occurrence of hallucinatory behavior.

Further, in view that the medicine 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 through oral, parenteral, pulmonary or nasal routes. According to this The specific forms of the medicaments described in the first aspect of the invention may be administered in a manner suitable for these routes of administration.

Dosage forms suitable for oral administration are capable of rapid action and/or modified release of the medicament described in the first aspect of the invention, and include crystalline and/or amorphous and/or dissolved forms, such as tablets ( Uncoated or coated tablets, which for example have a coating that resists gastric juices or delays dissolution or indissolution, and is released according to the properties of the drug itself as described in the first aspect of the invention), tablets that break rapidly in the mouth dosage form, or tablets, films/lyophilisates, capsules (e.g., hard or soft capsules), sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols, or solutions.

Parenteral administration may be administered in a form that avoids an absorption step (e.g., intravenous, intraarterial, intracardiac, intraspinal, intralumbar, intraarticular) or includes both absorption (e.g., intramuscular, subcutaneous, intradermal, transdermal or intraperitoneally). Suitable administration forms for parenteral administration are in particular preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophilisates or sterile powders.

Suitable for another route of administration are pharmaceutical forms for inhalation, for example powder inhalation or nebulizer inhalation, or pharmaceutical forms for nasal administration, infusion drops, solutions or sprays.

The medicament according to the first aspect of the invention can be converted into the dosage form. This can be carried out in a manner known per se by mixing with inert, non-toxic, pharmacologically suitable excipients. These excipients include in particular carriers (eg microcrystalline cellulose, lactose, mannitol, starch), solvents (eg liquid polyethylene glycols), emulsifiers and dispersants or wetting agents (eg dodecane sodium sulfate, polyoxysorbitan oleate, propylene glycol), binders (e.g., polyvinylpyrrolidone), synthetic and natural polymers (e.g., albumin), stabilizers (e.g., antioxidants, e.g. ascorbic acid), colorants (e.g., inorganic pigments, iron oxides), and masking fragrances and odors.

In addition, the present invention also provides Rho kinase activators for the preparation and treatment of analgesia, treatment of addiction, treatment of autoimmune diseases, improvement of depression and anxiety, Alzheimer's disease and related dementias, post-traumatic stress disorder, and autoimmune diseases. Application in drugs for psychiatric disorders such as autism spectrum disorders. In a specific embodiment of the invention, the Rho kinase activator is preferably LPA.

In a second aspect, the present invention provides pharmaceutical compositions for specifically inhibiting hallucinogenic effects.

Further, the pharmaceutical composition contains an effective amount of a Rho kinase inhibitor.

Further, the Rho kinase inhibitor is Fasudil.

Further, the pharmaceutical composition also contains one or more pharmaceutically acceptable inert, non-toxic excipients.

In the present invention, the term "effective amount" refers to the amount that has a therapeutic effect or the amount required to produce a therapeutic effect in a treatment subject. For example, a therapeutically or pharmaceutically effective amount of a drug is the amount of drug required to produce the desired therapeutic effect, which may be reflected by the results of clinical trials, model animal studies, and/or in vitro studies. The pharmaceutically effective dose depends on several factors, including but not limited to the characteristics of the treatment subject (such as height, weight, gender, age and medication history) and the severity of the disease.

Furthermore, the pharmaceutical composition may also contain other therapeutic agents for inhibiting or reducing hallucinations.

Further, the other therapeutic agents for suppressing or alleviating hallucinations include, but are not limited to: any currently disclosed agents that can be used to inhibit or reduce hallucinations, and/or any currently disclosed agents that can be used to assist in suppressing or alleviating hallucinations. The pharmaceutical composition composed of the above reagent and the Rho kinase inhibitor Fasudil of the present invention is also within the protection scope of the present invention. When the reagent is used in combination with the Rho kinase inhibitor Fasudil of the present invention, It can be used simultaneously or sequentially (use the reagent first and then use the Rho kinase inhibitor Fasudil of the present invention, or use the Rho kinase inhibitor Fasudil of the present invention first and then use the reagent). In addition, the above When the above reagent is used in combination with the Rho kinase inhibitor Fasudil of the present invention, the administration methods may be the same or different. The administration methods include but are not limited to: intravenous, subcutaneous, intraperitoneal, intracranial, and intrathecal. , intraarterial (for example, via the carotid artery), intramuscular, intranasal, or a combination of any of these.

Further, suitable administration methods of the pharmaceutical composition include any of various methods and delivery systems known to those skilled in the art to physically introduce the pharmaceutical composition of the present invention into the body of the subject, so The modes of administration include, but are not limited to: oral administration, parenteral administration, administration by inhalation spray, topical administration, rectal administration, nasal administration, buccal administration, vaginal administration or via implanted Drug storage device for administration. In specific embodiments of the present invention, injection administration is preferred, and injection administration includes rapid injection or continuous infusion. Where the pharmaceutical composition is for injection administration, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents such as suspending agents, preservatives, stabilizers and/or or dispersant.

In addition, the present invention also provides a method for inhibiting or reducing hallucinations, the method comprising the steps of: administering an effective amount of the Rho kinase inhibitor Fasudil or the second aspect of the present invention to a subject in need pharmaceutical compositions.

In a specific embodiment of the present invention, the hallucination effect is preferably a hallucination effect mediated by 5HT _2A receptors, that is, the hallucination effect induced by classic hallucinogens acting on 5-HT receptors and activating 5-HT2A receptors .

Description of drawings

Figure 1 shows the results of changes in Nogo-A and RhoA protein expression in the cerebral cortex of mice after intraperitoneal administration of the classic hallucinogens DOM and Psilocin, and the non-hallucinogenic 5-HT _2A receptor agonist Lisuride and TBG;

Figure 2 shows the results of phosphorylation changes of Nogo-A and RhoA proteins in the cerebral cortex after intraperitoneal administration of the classic hallucinogens DOM and Psilocin, the non-hallucinogenic 5-HT _2A receptor agonist Lisuride and TBG to mice;

Figure 3 shows the effect of Fasudil on the head shaking behavior of mice induced by the classic hallucinogen DOM, where n=8-10 for each group, data are expressed as mean ± SEM, *P<0.05, **P<0.01 , ***P<0.001, ****P<0.0001, data were analyzed using one-way ANOVA and Dunnett's test;

Figure 4 shows the effect of Fasudil on the head shaking behavior of mice induced by the classic hallucinogen Psilocin, where n=8-10 for each group, data are expressed as mean ± SEM, *P<0.05, **P<0.01 , ***P<0.001, ****P<0.0001, data were analyzed using one-way ANOVA and Dunnett's test;

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

Detailed ways

The present invention will be further described below with reference to specific examples, which are only used to explain the present invention and cannot be understood as limiting the present invention. Those of ordinary skill in the art can understand that various changes, modifications, substitutions and modifications can be made to these embodiments without departing from the principles and purposes of the invention. The scope of the invention is defined by the claims and their equivalents. . Experimental methods without specifying specific conditions in the following examples are usually tested according to conventional conditions or according to the conditions recommended by the manufacturer.

Example 1 Western Blot to detect changes in Nogo-A and RhoA after the action of hallucinogens 1. Experimental materials

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

Table 1 Experimental reagents

Table 2 Experimental antibodies

Table 3 Experimental instruments

The main reagent configurations in this example are:

10× running buffer of electrophoresis solution: Configure the electrophoresis solution: double distilled water at a ratio of 1:9.

10×electrotransfer transfer buffer: Configure the electrotransfer solution: methanol: double distilled water in a ratio of 1:2:7.

10×TBST: Configure TBST:double distilled water in a ratio of 1:9.

5% skimmed milk powder: Take 2g of skimmed milk powder and add 40mL of 1×TBST to fully dissolve.

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

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

2. Western Blot detects changes in Nogo-A and RhoA after the effects of hallucinogens

The samples used in this example are the classic hallucinogens DOM (1mg/kg) and Psilocin (0.5mg/kg) and the non-hallucinogenic 5-HT _2A receptor agonist Lisuride (T61065-Shanghai Taoshu Biotechnology Co., Ltd. ) (0.1mg/kg) and TBG (synthesized by the Institute of Toxicology and Drugs, Academy of Military Medical Sciences) (20mg/kg) were administered intraperitoneally to mice (Speifford (Beijing) Biotechnology Co., Ltd., C57, male, 18-22g, Protein extract of cerebral cortex at 10 min after 6-8 weeks). Western Blot experiment was used to detect changes in the expression levels of Nogo-A and RhoA proteins in the above samples. The specific Western Blot experimental methods are as follows:

(1) Glue making

①Choose a glass plate with a smooth lower edge, clean the glass plate and comb, rinse with distilled water and blow dry.

② Align the thick and thin glass plates and put them into the clamp to clamp them vertically on the shelf. During operation, the lower edges of the two glass plates must be aligned to avoid glue leakage.

③ Prepare the required separation gel according to the polyacrylamide gel formula, add TEMED and shake immediately to pour the gel. The formula of 10% separating gel is shown in Table 4 below, and the formula of 5% stacking gel is shown in Table 5 below.

Table 4 10% separating gel formula

Table 5 5% concentrated gel formula

(2) Glue filling and sample loading

① When filling glue, use a gun to add it 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 after liquid sealing will gel faster. When pouring glue, you can start faster and slow down when the glue surface reaches the required height. The glue must be applied very slowly and evenly from left to right, otherwise the glue will be washed out and deformed.

② When there is a fold line between the water and the glue (about 20 minutes at room temperature), the glue has solidified. Wait another 3 minutes for the glue to fully solidify, then pour off the upper water and blot it dry with filter paper.

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

④After gelation, rinse the gel with distilled water and put it into the electrophoresis tank. The thin glass panes are on the inside and the thick glass panes are on the outside. If only one gel is to be run, a plastic plate instead of a glass plate should be placed on the other side of the electrophoresis tank.

⑤ Fill the inner tank with new electrophoresis solution and prepare to load the sample. The electrophoresis solution in the inner tank must at least cover the inner glass plate, and the electrophoresis solution in the outer tank must be about 3cm high to cover the lower edge of the glass plate. Hold both sides of the comb with both hands and gently pull it out vertically. Use a 1 mL pipette to blow and rinse the sampling hole. To add samples, use a pipette to absorb the sample and insert the tip of the gun into the two plates above the sampling hole. Slowly add sample into the gap. The unsampled wells need to be filled with 1× loading buffer.

(3)Electrophoresis

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 electrophoresis. Electrophoresis is terminated when the bromophenol blue reaches the lower edge of the gel.

(4)Transfer film

① Prepare a piece of PVDF film whose area is slightly larger than the area of the adhesive surface to be transferred. To transfer a piece of glue, you need 8 pieces of thin filter paper or 4 pieces of thick filter paper (8×10cm). If it is a PVDF membrane, it needs to be activated with methanol for 30-60 seconds before use and the formula of the transfer solution contains methanol.

②Put the clamp for membrane transfer, two sponges, filter paper and membrane into a large glass dish filled with electrotransfer solution. Open the clamp so the black side is level. Place a piece of sponge paper on top and press the sponge with your hands to drive out the air bubbles and allow the electroconversion fluid to soak into the sponge. Place two layers of thick filter paper on the mat, fix the filter paper with one hand and remove the air bubbles with the other hand.

③Pry off the glass plate and peel it off, cut off the glue deformed by extrusion at the lower edge, and peel off the concentrated glue. Carefully peel off the separation gel and cover it on the filter paper, gently remove the air bubbles with your hands, cover the membrane on the gel, and do not move it after the cover is removed. Cover it with filter paper, sponge, and clamp it.

④Put the clip into the electric transfer cell, glue it on the negative electrode, and put the film on 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.

⑤After the transfer, use tweezers to take out the membrane, rinse it once with TBST, and pour it from the side to prevent the protein from being washed away.

(5) Antibody incubation

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

② Dilute the primary antibody according to a certain proportion with 5% BSA. Add primary antibody dilution and shake at room temperature for 1-4 hours or overnight at 4°C.

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

④ Use 5% milk to dilute the secondary antibody according to a certain ratio (usually 1:5000), add the secondary antibody diluent, and incubate for 1 hour on a shaker at room temperature.

⑤ Discard the secondary antibody and wash with TBST 3-4 times, 5 minutes each time.

(6)Develop

① Place the membrane protein side up on the blackboard, mix developer A/B solution 1:1 (change the pipe tip), then drop it on the membrane to fully cover it.

②Put the blackboard into the exposure machine, shoot the marker under white light conditions, and expose the target strip under self-illumination conditions.

(7)Data statistics

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

3. Experimental results

The classic hallucinogens DOM (1mg/kg) and Psilocin (0.5mg/kg), and the non-hallucinogenic 5-HT _2A receptor agonist Lisuride (0.1mg/kg) and TBG (20mg/kg) were intraperitoneally administered to mice. The results of the changes in the expression levels of Nogo-A and RhoA proteins in the protein extracts of the cerebral cortex at 10 minutes are shown in Figure 1. The results show that the changes in the Nogo-A protein are not obvious, and the amount of RhoA protein is increased after the administration of DOM and Psilocin. trend. This indicates that the RhoA protein may play a role in the acute effects of classic hallucinogens by changing the protein amount, while the Nogo-A protein may not play a role by changing the protein amount, but may play a role through protein modification or other forms of changes. .

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

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

Table 6 Experimental reagents

Table 7 Experimental antibodies

Table 8 Experimental instruments

Main reagent configurations in this example:

1×Electrophoresis solution running buffer: Configure the electrophoresis solution: double-distilled water at a ratio of 1:9.

1×electrotransfer transfer buffer: Configure the electrotransfer solution: methanol: double distilled water in a ratio of 1:2:7. 1×TBST: Configure TBST:double distilled water in a ratio of 1:9.

5% skimmed milk powder: Take 2g of skimmed milk powder and add 40mL of 1×TBST to fully dissolve.

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

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

Phosbind: 10mg Phosbind is dissolved in 0.10mL methanol and 3.2mL distilled water.

10mmol/L Mncl ²⁺ : 10mg dissolved in 5mL distilled water.

10mmol/L EDTA: 350mg EDTA dissolved in 120mL electrotransfer solution.

2. Experimental methods

The samples used in this example are the classic hallucinogens DOM (1mg/kg) and Psilocin (0.5mg/kg) and the non-hallucinogenic 5-HT _2A receptor agonist Lisuride (T61065-Shanghai Taoshu Biotechnology Co., Ltd. ) (0.1mg/kg) and TBG (synthesized by the Institute of Toxicology and Drugs, Academy of Military Medical Sciences) (20mg/kg) were administered intraperitoneally to mice (Speifford (Beijing) Biotechnology Co., Ltd., C57, male, 18-22g, Protein extract of cerebral cortex at 10 min after 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 experimental methods for Phos-tag gel electrophoresis detection are as follows:

(1) Glue making

①Choose a glass plate with a smooth lower edge, clean the glass plate and comb, rinse with distilled water and blow dry.

② Align the thick and thin glass plates, put them into the clamp, and clamp them vertically on the shelf. During operation, the lower edges of the two glass plates must be aligned to avoid glue leakage.

③ Prepare the required separation gel according to the polyacrylamide gel formula, add TEMED and shake immediately to pour the gel. The formula of 10% separating gel is shown in Table 9 below, and the formula of 5% stacking gel is shown in Table 10 below.

Table 9 10% separating gel formula

Table 10 5% concentrated gel formula

(2) Glue filling and sample loading

① When filling glue, use a gun to add it 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 after liquid sealing will gel faster. When pouring glue, you can start faster and slow down when the glue surface reaches the required height. The glue must be applied very slowly and evenly from left to right, otherwise the glue will be washed out and deformed.

② When there is a fold line between the water and the glue (about 20 minutes at room temperature), the glue has solidified. Wait another 3 minutes for the glue to fully solidify, then pour off the upper water and blot it dry with filter paper.

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

④After gelation, rinse the gel with distilled water and put it into the electrophoresis tank. The thin glass panes are on the inside and the thick glass panes are on the outside. If only one gel is to be run, a plastic plate instead of a glass plate should be placed on the other side of the electrophoresis tank.

⑤ Fill the inner tank with new electrophoresis solution and prepare to load the sample. The electrophoresis solution in the inner tank must at least cover the inner glass plate, and the electrophoresis solution in the outer tank must be about 3cm high to cover the lower edge of the glass plate. Hold both sides of the comb with both hands and gently pull it out vertically. Use a 1 mL pipette to blow and rinse the sampling hole. To add samples, use a pipette to absorb the sample and insert the tip of the gun into the two plates above the sampling hole. Slowly add sample into the gap. The unsampled wells need to be filled with 1× loading buffer.

(3)Electrophoresis

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 electrophoresis. Electrophoresis is terminated when the bromophenol blue reaches the lower edge of the gel.

(4) Washing film

After electrophoresis and before transfer to membrane, chelating agent (EDTA) needs to be used to remove manganese ions (Mn ²⁺ ) from the gel. This step increases the transfer efficiency of phosphorylated and non-phosphorylated proteins onto PVDF membranes.

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

② Soak the gel in ordinary transfer buffer without EDTA for 10 minutes while shaking gently (10 minutes × 1 time).

(5)Transfer film

① Prepare a piece of PVDF film whose area is slightly larger than the area of the adhesive surface to be transferred. To transfer a piece of glue, you need 8 pieces of thin filter paper or 4 pieces of thick filter paper (8×10cm). If it is a PVDF membrane, it needs to be activated with methanol for 30-60 seconds before use and the formula of the transfer solution contains methanol.

②Put the clamp for membrane transfer, two sponges, filter paper and membrane into a large glass dish filled with electrotransfer solution. Open the clamp so the black side is level. Place a piece of sponge paper on top and press the sponge with your hands to drive out the air bubbles and allow the electroconversion fluid to soak into the sponge. Place two layers of thick filter paper on the mat, fix the filter paper with one hand and remove the air bubbles with the other hand

③Pry off the glass plate and peel it off, cut off the glue deformed by extrusion at the lower edge, and peel off the concentrated glue. Carefully peel off the separation gel and cover it on the filter paper, gently remove the air bubbles with your hands, cover the membrane on the gel, and do not move it after the cover is removed. Cover it with filter paper, sponge, and clamp it.

④Put the clip into the electric transfer cell, glue it on the negative electrode, and put the film on 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.

⑤After the transfer, use tweezers to take out the membrane, rinse it once with TBST, and pour it from the side to prevent the protein from being washed away.

(6) Antibody incubation

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

② Dilute the primary antibody according to a certain proportion with 5% BSA. Add primary antibody dilution and shake at room temperature for 1-4 hours or overnight at 4°C.

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

④ Use 5% milk to dilute the secondary antibody according to a certain ratio (usually 1:5000), add the secondary antibody diluent, and incubate for 1 hour on a shaker at room temperature.

⑤ Discard the secondary antibody and wash with TBST 3-4 times, 5 minutes each time.

(7)Develop

① Place the membrane protein side up on the blackboard, mix developer A/B solution 1:1 (change the pipe tip), then drop it on the membrane to fully cover it.

②Put the blackboard into the exposure machine, shoot the marker under white light conditions, and expose the target strip under self-illumination conditions.

(8)Data statistics

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

3. Experimental results

According to the principle of Phos-Tag experiment, if a protein has two closely spaced bands, the upper band represents the amount of protein that has undergone phosphorylation modification, and the lower band represents the amount of protein that has not undergone phosphorylation modification. The upper and lower bands represent the total protein amount of the protein.

Detection of the classic hallucinogens DOM (1mg/kg) and Psilocin (0.5mg/kg), and the non-hallucinogenic 5-HT2A receptor agonist Lisuride (0.1mg/kg) and TBG (20mg/kg) intraperitoneally administered to mice Finally, the results of the phosphorylation changes of Nogo-A and RhoA proteins in the protein extract sample of the mouse cerebral cortex at 10 minutes are shown in Figure 2. The results show that only one band of Nogo-A protein was detected. The phosphorylation changes were not obvious. Two bands were detected for RhoA protein. The upper band is the amount of phosphorylated protein, and the lower band is the amount of protein that has not undergone phosphorylation changes. It can be seen that the phosphorylation changes of RhoA in the Psilocin group showed a downward trend. This indicates that RhoA protein may play a role in the acute effects of classic hallucinogens by changing the protein amount and undergoing phosphorylation modification. The Nogo-A protein may not function through phosphorylation modification.

Example 3 Study on the inhibitory effect of Fasudil on hallucinations induced by classic hallucinogens

1. Experimental materials

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

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

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

Table 11 Experimental reagents

Table 12 Experimental equipment

Main reagent configurations in this example:

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

Psilocin solution preparation: Weigh an appropriate amount of sample and dissolve it in 1% DMSO and 99% normal saline to prepare a solution of 0.5 mg/mL. Use normal saline to dilute it to 0.05 mg/mL, which is suitable for intraperitoneal injection in mice.

Preparation of Rho kinase (ROCK) inhibitor Fasudil solution: Weigh an appropriate amount of sample and dissolve it in physiological saline, prepare it to 2mM, and dilute it gradiently to 1mM and 0.2mM for injection into the lateral cerebral ventricle of mice.

Preparation of P75 NTR inhibitor TAT-Pep5 solution: Weigh an appropriate amount of sample and dissolve it in physiological saline, prepare it to 1 mg/mL, and dilute it gradiently to 0.2 mg/mL and 0.04 mg/mL for injection into the lateral cerebral ventricle of mice.

2. Head-flick reaction experiment to verify the inhibitory effect of Fasudil on hallucinations induced by classic inhibitors

(1) Effect of Fasudil on hallucinations induced by classic inhibitors

The head toss response (HTR) is a rapid side-to-side rotation of the head that occurs after administration of serotonergic hallucinogens or other 5-HT _2A agonists to rats and mice. The head toss response is widely used as a 5 - Behavioral assay of HT _2A receptor activation. In classic psychedelic research, the head-flick response is by far the most commonly used animal model. There is a strong positive correlation between the head shaking reaction effect in mice and the hallucinogenic effect in humans, that is, the head shaking reaction can indicate the occurrence of hallucinatory behavior.

The experimental mice described in this example were first administered the above-mentioned tool drug (Fasudil) into the lateral cerebral ventricle. After waiting for 30 minutes, the classic hallucinogen DOM or Psilocin was injected intraperitoneally. Immediately after the injection, the animals were put into a transparent box to observe the head shaking reaction behavior and perform manual counting. The behavioral test was conducted for 15 or 30 minutes.

Among them, the dose of DOM is 1 mg/kg, which is injected by intraperitoneal injection; the dose of Psilocin is 0.5 mg/kg, which is injected by intraperitoneal injection. Three doses of the tool drug (Fasudil) were selected: low (1nmol/5μL), medium (5nmol/5μL), and high (10nmol/5μL). They were all injected by lateral ventricular injection 30 minutes before the administration of the hallucinogen. . The volume for intracerebroventricular injection is 5 μL, and the volume for intraperitoneal injection is 0.2 mL/20 g.

(2) Effect of P75 NTR inhibitor TAT-Pep5 on hallucinations induced by classic inhibitors

This example further verified 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, which was injected intraperitoneally. Three doses of TAT-Pep5 were selected: low (0.2 μg/5 μL), medium (1 μg/5 μL), and high (5 μg/5 μL), all of which were injected into the lateral cerebral ventricle 30 minutes before DOM administration.

3. Experimental results

The inhibitory effect of Fasudil on hallucinations induced by classic inhibitors is shown in Figures 3 and 4. The left pictures in Figures 3 and 4 show that Fasudil has a significant inhibitory effect on head shaking behavior, and shows a dose gradient change, indicating that Fasudil It can significantly inhibit the head shaking behavior of mice induced by classic hallucinogens, that is, Fasudil of the present invention has a significant inhibitory effect on the hallucinations induced by classic hallucinogens; the right pictures in Figures 3 and 4 show head shaking. The changes in the number of head flicks within 15 or 30 minutes were reflected, and the cumulative count was calculated every 5 minutes. The results showed that about 5 to 10 minutes after the administration of the hallucinogen, there was a significant difference in the number of head flicks between the Fasudil administration group and the control group. The above results further indicate that Fasudil can be used in the preparation of drugs that specifically inhibit hallucinations.

The results of the effect of the P75 NTR inhibitor TAT-Pep5 on the head-shaking behavior of mice induced by classic hallucinogens are shown in Figure 5. The left picture in Figure 5 shows the effects of low, medium and high doses of TAT-Pep on the head-shaking behavior of mice. There is no obvious inhibitory or up-regulating effect on behaviors, 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; the right picture in Figure 5 shows the head shaking experiment30 The change in the number of head flicks within minutes is counted once every 5 minutes. It can be seen that there is no obvious difference in the head flick number curves of the four groups. That is to say, not all proteins in the Nogo-A/RhoA signaling pathway can inhibit hallucinations.

The description of the above embodiments is only for understanding the method of the present invention and its core idea. It should be noted that those of ordinary skill in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims (10)

1. Use of a rho kinase inhibitor in the manufacture of a medicament for specifically inhibiting the hallucination.
2. 2. The use according to claim 1, wherein the Rho kinase inhibitor is Fasudil.
3. 3. The use according to claim 2, wherein the hallucinations are 5HT _2A Receptor mediated hallucination.
4. 4. The use according to claim 3, wherein the medicament further comprises one or more inert, non-toxic excipients that are pharmaceutically acceptable.
5. 5. The use according to claim 4, wherein the excipient comprises a carrier, a solvent, an emulsifier, a dispersant, a wetting agent, a binder, a stabilizer, a colorant, a perfume.
6. 6. The use according to claim 5, wherein the medicament is in the form of an injection, capsule, granule, drop, lyophilisate, granulate or tablet.
7. 7. The use according to claim 6, wherein the medicament is in the form of injection, capsule, granule, drop, freeze-dried product, granule or tablet by adding pharmaceutical auxiliary ingredient to Fasudil of 80% or more by mass fraction.
8. 8. A pharmaceutical composition for specifically inhibiting the hallucinations, said pharmaceutical composition comprising an effective amount of a Rho kinase inhibitor.
9. 9. The pharmaceutical composition of claim 8, wherein the Rho kinase inhibitor is Fasudil.
10. 10. The pharmaceutical composition of claim 9, further comprising one or more inert, non-toxic excipients that are pharmaceutically acceptable.