Use of trifluperidol in the treatment of cerebrovascular disorders

Abstract

The application belongs to the technical field of medicine, and particularly relates to application of trifluperidol in treatment of cerebrovascular diseases. Trifluperidol is a D1 / D2 dopamine receptor antagonist with oral activity and a novel PI3K inhibitor, and trifluperidol has anti-proliferative activity on cancer cells and induces apoptosis. The application discloses trifluperidol for the first time, and no report is found on effective treatment of trifluperidol against ischemic stroke, especially the application creatively proposes that trifluperidol significantly reduces the volume of acute cerebral infarction of a mouse with focal cerebral ischemia, which indicates that trifluperidol can be used for treating ischemic stroke.

Description

Technical Field

[0001] This invention pertains to pharmaceutical technology, specifically the application of trifluorothioxanol in the treatment of cerebrovascular diseases. Background Technology

[0002] Stroke is a group of diseases characterized by ischemic and hemorrhagic brain injury, also known as cerebrovascular accident. It has a rapid onset and extremely high rates of disability and death, making it the second leading cause of death worldwide, and this problem is becoming increasingly serious with an aging population. Acute ischemic stroke (acute cerebral infarction) is the most common type of stroke, accounting for 69.6% to 70.8% of strokes in my country.

[0003] The pathogenesis of ischemic stroke is complex, involving multiple mechanisms such as peroxidation, energy metabolism disorders, calcium overload, and excitatory amino acid toxicity. Currently, the only drug approved by the US FDA for the treatment of ischemic stroke is tissue plasminogen activator (t-PA, intravenously), which can dissolve thrombi, restore blood flow, and reopen blood vessels. However, due to the narrow therapeutic window of t-PA (effective 4.5 hours after stroke) and its tendency to cause bleeding, only a small percentage of patients receive thrombolytic therapy. Therefore, finding new targets for treating ischemic stroke, drugs with novel mechanisms of action and fewer adverse reactions, is an urgent problem to be solved. Summary of the Invention

[0004] This invention discloses the application of trifluorothioxol in the treatment of cerebrovascular diseases; it confirms that trifluorothioxol has a therapeutic effect on ischemic stroke, providing new targets, new mechanisms of action and new ideas for drug development for cerebrovascular diseases such as ischemic stroke.

[0005] The present invention adopts the following technical solution:

[0006] Application of trifluorothioxanol in the preparation of drugs for treating cerebrovascular diseases.

[0007] The use of trifluorothioxol in the preparation of drugs that reduce the volume of cerebral infarction. Preferably, the use of trifluorothioxol in the preparation of drugs that reduce the volume of cerebral infarction in the acute phase of stroke; more preferably, the use of trifluorothioxol in the preparation of drugs that reduce the volume of cerebral infarction in the acute phase of focal ischemic stroke.

[0008] Application of trifluorothioxanol in the preparation of drugs that inhibit reactive astrocyte proliferation.

[0009] The use of trifluorothioxanol in the preparation of drugs for improving neurological symptom scores. Preferably, the use of trifluorothioxanol in the preparation of drugs for improving neurological symptom scores during the acute phase of cerebral ischemia.

[0010] In this invention, the active ingredient of the drug is trifluorothioxanol or its salt, which can be used alone as a drug ingredient or combined with pharmaceutically acceptable excipients, such as dispersants, etc., which are conventional techniques; the drug dosage form is also conventionally selected, such as solution, ointment, suppository, tablet, powder, etc.

[0011] In this invention, the disease is an ischemic disease, specifically an ischemic cerebrovascular disease, such as ischemic stroke.

[0012] Trifluthixoxanol is an orally active D1 / D2 dopamine receptor antagonist and a novel PI3K inhibitor (PI3Kα IC50). 50 Trifluorothioxan (127 nM) exhibits anti-proliferative activity against cancer cells and induces apoptosis. This invention, for the first time, discloses an effective treatment of ischemic stroke using trifluorothioxan, a method previously unreported. This invention creatively proposes that trifluorothioxan significantly reduces the volume of acute cerebral infarction in mice with focal cerebral ischemia, demonstrating that trifluorothioxan can be used to treat ischemic stroke. Attached Figure Description

[0013] Figure 1 The inhibition curve for trifluorothioxanol is shown.

[0014] Figure 2 The figure shows the effect of trifluorothioxanol on reducing the volume of cerebral infarction in mice during the acute phase of cerebral ischemia.

[0015] Figure 3 This is a schematic diagram illustrating the effect of trifluorothioxanol on improving neurological symptoms in mice with cerebral ischemia.

[0016] Figure 4 This is a schematic diagram illustrating the effects of trifluorothioxanol on palmitoylated transferase DHHC5 and reactive astrocyte GFAP.

[0017] Figure 5 The figure shows the results of a comparative experiment of trifluorothioxanol, 2-bromohexadecanoic acid, and edaravone dextranol. Detailed Implementation

[0018] Trifluorothioxanol is an existing compound with the following chemical structural formula:

[0019] The reagents and raw materials involved in this invention are existing technologies, and the specific experimental methods and tests are conventional techniques. Animal experiments meet the relevant requirements of Soochow University. The dosage range for animal experiments can be 10 mg / kg to 30 mg / kg, and the drug is trifluorothioxanol, which is routinely dissolved in physiological saline.

[0020] Western blotting method.

[0021] 1. Protein sample preparation: Add RIPA lysis buffer (strong) (main components are 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100) to mouse cerebral cortex tissue, sonicate to disrupt cells, centrifuge at 12000 g for 15 min at 4℃, and transfer the supernatant to a new centrifuge tube.

[0022] 2. Protein concentration determination: After the protein was placed in a 37℃ incubator for 30 min, the absorbance was detected at 570 nm using an ELISA reader.

[0023] 3. Electrophoresis: Prepare a gel of the corresponding concentration based on the molecular weight of the protein being sampled, and add the prepared brain tissue or human astrocyte sample. Run the gel at 90V, and after gel running, transfer the gel to a membrane at 200 mA for 90 min.

[0024] 4. Immunoassay: After transfer, transfer the PVDF membrane to a petri dish containing blocking buffer (5% skim milk) and block on a shaker at room temperature for 1 h. Dilute the primary antibody with 1% BSA, add it to the sealed hybridization bag, remove air bubbles, seal, and incubate at room temperature for 3 h or overnight on a shaker at 4°C. Wash the membrane three times with TBST for 10 min each time, add fluorescent secondary antibody, and incubate at room temperature for 1 h. Wash the membrane three times with TBST for 10 min each time. Develop using an Odyssey infrared fluorescence scanning imaging system.

[0025] Example 1

[0026] Figure 1 The half-maximal inhibitory concentration (IC50) of trifluorothioxanol 50 Human astrocytes were treated with different concentrations of trifluorothioxanol for 24 hours, and the absorbance of the cells at 450 nm was measured using the CCK-8 assay. The IC50 values ​​were... 50 =9.363μM. Specific experimental procedure: After cell counting, 10 μL of the solution was applied to each well. 3 Cells were seeded into 96-well plates at 100 μl per well. After cell attachment, trifluthixoxanol was diluted with physiological saline to different concentrations, with 5 replicates per group. Cell proliferation was detected after 24 h of incubation. 10 μl of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance at 450 nm was measured using a microplate reader. The cell inhibition rate was calculated for each group. The drug concentration at which 50% inhibition was achieved was plotted on the x-axis as the drug concentration and the inhibition rate on the y-axis. 50 .

[0027] Example 2

[0028] Effects of trifluthixol on ischemic stroke. Male C57 mice were randomly divided into a control group (sham), a model group (I / R), and a treatment group (I / R + trifluthixol), with 10 mice in each group. A mouse model of transient middle cerebral artery occlusion (tMCAO) was established using the suture occlusion method. After 60 min of ischemia, reperfusion was performed, and trifluthixol (20 mg / kg) was immediately administered intraperitoneally during reperfusion, as a single dose. Twenty-four hours later, the mice were decapitated, and brain sections were collected and stained with TTC to observe the effect of trifluthixol on the infarct volume during the acute phase of cerebral ischemia. Before sacrifice, the Longa method was used to score the neurological symptoms of mice after reperfusion (I / R) during the acute phase of cerebral ischemia. The higher the score, the more severe the neurological deficit. Table 1 shows the neurological symptom scoring criteria.

[0029]

[0030] Figure 2 The figure shows the effect of trifluorothioxan on reducing the infarct volume in mice with acute cerebral ischemia. As can be seen from the figure, trifluorothioxan can reduce the infarct volume in mice with acute cerebral ischemia (p<0.0001, which is extremely significant).

[0031] Figure 3 This is a schematic diagram illustrating the effect of trifluorothioxanol on improving neurological symptoms in mice with cerebral ischemia.

[0032] Figure 4 This diagram illustrates the effects of trifluorothioxanol on palmitoylated transferase DHHC5 and reactive astrocyte GFAP. As shown in the figure, the model group significantly increased the expression of DHHC5 and GFAP, while trifluorothioxanol effectively inhibited the expression of palmitoylated transferase DHHC5 and reactive astrocyte GFAP.

[0033] Existing technology considers trifluorothioxan to be an orally active D1 / D2 dopamine receptor antagonist and a novel PI3K inhibitor, possessing anti-proliferative activity against cancer cells and inducing apoptosis. This invention, for the first time, discloses trifluorothioxan's effective treatment of ischemic stroke, an efficacy previously unreported. This invention discloses that trifluorothioxan can effectively inhibit the expression of palmitoylated transferase DHHC5 and reactive astrocyte GFAP, significantly reducing the volume of acute cerebral infarction in mice with focal cerebral ischemia, and can be used to treat ischemic stroke.

[0034] Example 3

[0035] Male C57 mice were randomly divided into a model group (I / R) and drug treatment groups (I / R + 2-bromohexadecanoic acid 20 mg / kg, I / R + edaravone dexborneol 10 mg / kg, I / R + trifluthixoxanol 10 mg / kg), with 10 mice in each group. A transient middle cerebral artery occlusion (tMCAO) model was established in mice using the suture occlusion method. After 60 min of ischemia, reperfusion was performed, with the drugs administered intraperitoneally immediately upon reperfusion, as a single dose. Changes in DHHC5 levels in the brain were observed 24 hours later. (See [link to relevant documentation]). Figure 5 It is known that trifluorothioxan has the best effect compared with existing palmitoylation inhibitors such as 2-bromohexadecanoic acid and edaravone dextranol, a commonly used cerebrovascular disease drug in clinical practice.

[0036] Therefore, trifluorothioxan significantly inhibits DHHC5 palmitoylation transferase, reduces the volume of acute cerebral infarction in mice with focal ischemic stroke, and significantly improves their neurobehavioral symptoms. Trifluorothioxan can be considered a promising drug for treating cerebrovascular diseases, and as a general knowledge, it can also be used as an active ingredient in conventional combinations with pharmaceutical excipients. The excipients in this invention can be varied depending on the dosage form, administration method, etc. Excipients include excipients, binders, disintegrants, lubricants, flavoring agents, fragrances, colorants, and sweeteners. The drug can be administered orally, sublingually, transdermally, intramuscularly or subcutaneously, through the skin or mucous membrane, or intravenously. The drug can be in conventional pharmaceutical formulations such as capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalers, creams, ointments, suppositories, or patches.

Claims

1. The use of trifluorothioxanol or a pharmaceutically acceptable salt thereof in the preparation of drugs for treating ischemic stroke, characterized in that, The dosage form of the drug is capsules, granules, or pills.

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