Use of CTMB in preparation of drugs for treating and / or preventing ischemic stroke

Abstract

The application provides application of CTMB in preparation of a medicine for treating and / or preventing ischemic cerebral stroke, a milk injection for treating ischemic cerebral stroke and a preparation method of the milk injection, and provides a medicine for treating or preventing ischemic cerebral stroke, which can significantly improve the neurological behavior and sensory and motor functions of a model rat of ischemic cerebral stroke, is effective and safe, and has no obvious toxic side effects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and specifically relates to the application of CTMB in the preparation of drugs for the treatment and / or prevention of ischemic stroke. Background Technology

[0002] Stroke has become the second leading cause of death and the third leading cause of disability worldwide, with more than 80% of cases being ischemic stroke. Ischemic stroke, also known as cerebral infarction, is brain damage caused by localized or complete restriction of blood flow to the brain. It is characterized by high incidence, high mortality, high disability rate, and high recurrence rate. Ischemic stroke can lead to severe clinical symptoms such as hemiplegia, strabismus, aphasia, sensory disturbances, and decreased consciousness, significantly impacting quality of life and placing a huge burden on families and society.

[0003] The development of ischemic stroke involves a complex multi-stage pathological mechanism, including oxidative stress damage, mitochondrial dysfunction, apoptosis, inflammatory responses, and blood-brain barrier damage. During ischemic stroke, the interruption of blood supply to the brain leads to hypoglycemia and hypoxia, causing ATP depletion in cells, impaired energy-dependent ion transport, depolarization of neurons and glial cells, and calcium ion influx leading to the release of excitatory amino acids such as glutamate, resulting in excitotoxic effects. The large amount of calcium ions in cells activates metabolic enzymes, producing large amounts of nitric oxide, arachidonic acid metabolites, and oxygen free radicals, inducing oxidative stress damage in the brain, which in turn triggers neuronal apoptosis and necrosis. While restoring cerebral blood flow quickly can alleviate ischemic brain damage, reperfusion exceeding a certain time limit may worsen brain injury. This is because reperfusion further increases free radical generation, exacerbating neuronal death. Necrotic cells release damage-associated pattern molecules (DAMPs), which promote chemotaxis of inflammatory cells and increase the production of inflammatory factors, chemokines, and ROS, leading to immune cell infiltration and exacerbating cerebral ischemia and blood-brain barrier disruption. On the other hand, inflammatory factors activate matrix metalloproteinases in the brain, increasing blood-brain barrier permeability, worsening the condition of stroke patients, and severely impacting their prognosis.

[0004] Currently, commonly used drugs for the clinical treatment of ischemic stroke mainly include recombinant tissue plasminogen activator (rt-PA) and neuroprotective agents. rt-PA is the most commonly used drug for treating ischemic stroke. However, rt-PA has a narrow clinical window and may increase the risk of cerebral hemorrhage and induce adverse reactions. Neuroprotective agents can reduce neuronal death after cerebral ischemia, improve the tolerance of nerve cells to ischemia and hypoxia, promote the recovery of neurological function, and improve patient prognosis. Currently used neuroprotective agents mainly include free radical scavengers such as edaravone and GABA receptor agonists such as piracetam. However, most marketed neuroprotective agents are single-target drugs and often act on the mid-to-downstream links of secondary neurological damage caused by stroke, limiting their clinical efficacy. Therefore, developing new drugs that can effectively treat ischemic stroke has significant clinical value and social significance. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to avoid the shortcomings of the prior art and provide the application of CTMB in the preparation of drugs for the treatment and / or prevention of ischemic stroke, which significantly improves the neurobehavioral and sensorimotor functions of rats with ischemic stroke, and is an effective, safe drug without obvious toxic side effects.

[0006] The above-mentioned objectives of the present invention are achieved through the following technical measures:

[0007] This invention provides the use of CTMB in the preparation of drugs for the treatment and / or prevention of ischemic stroke, wherein the structural formula of CTMB is shown in Formula 1.

[0008]

[0009] Preferably, the route of administration of the drug includes injection, oral administration, transdermal administration, inhalation, mucosal administration, or subcutaneous implantation.

[0010] Preferably, the drug is an injectable preparation.

[0011] Preferably, the injectable is an emulsion injection.

[0012] The present invention also provides an emulsion injection for treating ischemic stroke, comprising the following components by weight percentage: CTMB 0.5%–5%, oil phase 5%–30%, emulsifier 0.6%–1.8%, pH adjuster 0.001%–0.01%, and the balance being water.

[0013] Preferably, the emulsion injection further contains 0% to 2.5% glycerol.

[0014] Preferably, the oil phase is selected from one or more of soybean oil, medium-chain triglycerides, fish oil, olive oil, and structured triglycerides;

[0015] The emulsifier is selected from one or more of egg yolk lecithin, soybean lecithin, Pluronic F 68, and polyethylene glycol stearic acid-15 (Solutol HS15).

[0016] Preferably, the effective dosage of the active ingredient CTMB in the emulsion injection is in the ratio of 0.2 mg to 4.0 mg / kg of human body mass;

[0017] The effective dosage of the active ingredient CTMB in the emulsion injection, in ratio to rat unit mass, is 10 mg to 20 mg / kg.

[0018] The present invention also provides a method for preparing the emulsion injection, characterized by comprising the following steps:

[0019] (1) Under nitrogen or inert gas protection, CTMB is dissolved in an oil phase preheated to 70-80°C, and then the emulsifier is dissolved in the oil phase in which CTMB is dissolved or in an aqueous phase at 70-80°C.

[0020] (2) The oil phase and aqueous phase prepared above are mixed by high-speed shearing to prepare a primary emulsion, and the pH value is adjusted.

[0021] (3) The colostrum was homogenized under high pressure 1 to 3 times until the average droplet size was ≤0.4μm, filtered, and sterilized by rotary autoclaving to obtain an emulsion injection containing CTMB.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The drug prepared using CTMB can significantly improve the neurobehavioral function of rats with ischemic stroke and restore their sensory and motor functions.

[0024] (2) Effective and safe, with no obvious toxic side effects. Detailed Implementation

[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] This invention provides the use of CTMB in the preparation of drugs for the treatment and / or prevention of ischemic stroke, wherein the structural formula of CTMB is shown in Formula 1.

[0027]

[0028] In this invention, the routes of administration of the drug include injection, oral administration, transdermal administration, inhalation, mucosal administration, or subcutaneous implantation.

[0029] In this invention, the drug is preferably an injection, and more preferably an emulsion injection.

[0030] The present invention also provides an emulsion injection for treating ischemic stroke, comprising the following components by weight percentage: CTMB 0.5%–5%, oil phase 5%–30%, emulsifier 0.6%–1.8%, pH adjuster 0.001%–0.01%, and the balance being water.

[0031] In this invention, the emulsion injection solution comprises 0.5% to 5% CTMB, preferably 0.5% to 2%, more preferably 0.5%;

[0032] In this invention, the emulsion injection comprises 5% to 30% oil phase, preferably 10% to 20%, more preferably 10%, wherein the oil phase is selected from one or more of soybean oil, medium-chain triglycerides, fish oil, olive oil, and structured triglycerides;

[0033] The emulsion injection solution described in this invention comprises 0.6% to 1.8% emulsifier, preferably 0.6% to 1.5%, more preferably 1.2%; the emulsifier is selected from one or more of egg yolk lecithin, soybean lecithin, Pluronic F 68, and polyethylene glycol stearic acid-15 (Solutol HS15);

[0034] In this invention, the emulsion injection solution includes a pH adjuster of 0.001% to 0.01%, preferably 0.005%; the pH adjuster is a pharmaceutically acceptable base, selected from one or more of sodium hydroxide, sodium carbonate, and sodium bicarbonate;

[0035] In this invention, the emulsion injection solution includes water, preferably water for injection;

[0036] In this invention, the emulsion injection preferably further contains 0% to 2.5% glycerol, more preferably 2.0% to 2.5%, and even more preferably 2.25%, whereby glycerol acts as an osmotic pressure regulator;

[0037] In this invention, the effective dosage of the active ingredient CTMB in the emulsion injection is 0.2 mg to 4.0 mg / kg of human body mass; the effective dosage of the active ingredient CTMB in the emulsion injection is 10 mg to 20 mg / kg of rat body mass.

[0038] This invention also provides a method for preparing an emulsion injection, comprising the following steps:

[0039] (1) Under nitrogen or inert gas protection, CTMB is dissolved in an oil phase preheated to 70-80°C, and then the emulsifier is dissolved in the oil phase in which CTMB is dissolved or in an aqueous phase at 70-80°C.

[0040] (2) The oil phase and aqueous phase prepared above are mixed by high-speed shearing to prepare a primary emulsion, and the pH value is adjusted.

[0041] (3) The colostrum was homogenized under high pressure 1 to 3 times until the average droplet size was ≤0.4μm, filtered, and sterilized by rotary autoclaving to obtain an emulsion injection containing CTMB.

[0042] In this invention, in step (1), CTMB is dissolved in an oil phase preheated to 70-80°C, preferably 78°C;

[0043] In this invention, in step (1), the emulsifier is dissolved in the oil phase or the aqueous phase at 70-80°C, preferably at 78°C.

[0044] In this invention, in step (1), the preferred method of dissolving is to dissolve by stirring;

[0045] In this invention, the high-speed shearing time in step (2) is preferably 5 to 15 minutes, more preferably 10 minutes;

[0046] In this invention, the high-speed shearing speed in step (2) is preferably 13000 rpm;

[0047] In this invention, the pH value is preferably adjusted using sodium hydroxide in step (2);

[0048] In this invention, the pH value is preferably adjusted to 8.5 to 11.0 in step (2), and more preferably to 10.5;

[0049] In this invention, the high-pressure homogenization pressure in step (3) is preferably 800 bar;

[0050] In this invention, in step (3), the colostrum is homogenized under high pressure 1 to 3 times, preferably 2 times;

[0051] In this invention, the filtration in step (3) is preferably performed using a filter with a pore size of 5 μm; in this invention, the sterilization in step (3) is preferably performed by rotary autoclaving, with a temperature of 121°C, a time of 12 min, and a pressure of 103.4 kPa.

[0052] Example 1

[0053] Preparation of CTMB

[0054] 1.1 Raw materials used:

[0055] Cyclobutylcarboxylic acid (C13062789, Shanghai Maclean Biochemical Technology Co., Ltd.)

[0056] Oxaloyl chloride (JHSOWRS, Shanghai Xianding Biotechnology Co., Ltd.)

[0057] Dichloromethane (OPRN3RFE, Anhui Zesheng Technology Co., Ltd.)

[0058] 2,4,5-Trimethoxybenzaldehyde (T819646, Shanghai Maclean Biochemical Technology Co., Ltd.)

[0059] Aluminum trichloride (YZE8RSRV, Saas Chemical Technology (Shanghai) Co., Ltd.)

[0060] Sodium borohydride (2018041701, Chengdu Kelong Chemical Reagent Factory)

[0061] Tetrahydrofuran (T818767, Shanghai Maclean Biochemical Technology Co., Ltd.)

[0062] Acetic anhydride (2021123101, Chengdu Kelon Chemicals Co., Ltd.)

[0063] Anhydrous copper sulfate (C10843174, Shanghai Maclean Biochemical Technology Co., Ltd.)

[0064] Anhydrous sodium acetate (Tianjin Kemio Chemical Reagent Co., Ltd.)

[0065] Anhydrous magnesium sulfate (Q / 12KM3936-2019, Tianjin Kemeo Chemical Reagent Co., Ltd.)

[0066] Silicone sheet (10052521046809, Qingdao Ocean Chemical Co., Ltd.).

[0067] 1.2 Preparation process: Cyclobutylcarboxylic acid was dissolved in 150 mL of anhydrous dichloromethane under N2 protection in a three-necked flask and stirred at room temperature. Oxaloyl chloride was gradually added dropwise, and the mixture was stirred at room temperature until no bubbles were generated. The solution was concentrated to obtain 25.0 g of orange-yellow intermediate liquid. The liquid obtained above was dissolved in 100 mL of anhydrous dichloromethane, aluminum trichloride was added at 0 °C, the temperature was raised to room temperature, and the mixture was stirred for 2.5 hours. Then, 300 mL of water was added to quench the reaction, and the mixture was extracted with ethyl acetate (3 × 200 mL). The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated to obtain the crude product. The crude product was purified by slurrying with 40 mL of ethanol for 1 hour to obtain 43.7 g of white solid. The solid obtained above was dissolved in 150 mL of tetrahydrofuran. 20 mL of sodium borohydride aqueous solution and 10 drops of 10% sodium hydroxide solution were added dropwise at 0 °C. The temperature was raised to 60 °C and stirred for 3 hours. After cooling to 37 °C, the pH was adjusted to 7–8 with 1N hydrochloric acid solution. The tetrahydrofuran was removed by concentration, and the mixture was extracted with ethyl acetate (3 × 200 mL). The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated to obtain 42.6 g of an orange-yellow semi-solid (compound 1f). Anhydrous sodium acetate (8.31 g, 101.25 mmol) was added and dissolved in 210 mL of acetic anhydride. The temperature was raised to 140 °C and heated for 3 hours. The acetic anhydride was removed by concentration, and 200 mL of water was added. The mixture was extracted with ethyl acetate (4 × 200 mL). The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated to obtain the crude product. The crude product was purified by recrystallization from 70% ethanol to obtain 23.47 g of CTMB as a white solid, yield: 59.4%.

[0068] Nuclear magnetic resonance (NMR) results: 1H-NMR (400MHz, CDCl3) δ 6.82 (s, 1H), 6.51 (s, 1H), 6.34 (t, J = 2.4Hz, 1H), 3.88 (s, 3H), 3.82 (s, 3H), 3.81 (s, 3H), 3.13–2.95 (m, 2H), 2.89 (d, J = 6.1Hz, 2H), 2.09 (p, J = 7.8Hz, 2H).

[0069] 13C-NMR (100MHz, CDCl3) δ 150.7, 148.0, 143.1, 142.5, 119.0, 114.5, 111.4, 98.0, 56.9, 56.6, 56.2, 32.8, 32.6, 18.5.

[0070] Example 2

[0071] Preparation of CTMB emulsion injection (abbreviated as TK-X07)

[0072] 2.1 Experimental Materials:

[0073] CTMB [1-(cyclobutyrylmethyl)-2,4,5-trimethoxybenzene] (prepared in Example 1),

[0074] Soybean oil for injection (DD20200603, Shandong Ruisheng Pharmaceutical Excipients Co., Ltd.),

[0075] Egg yolk lecithin (202008013, Shanghai Taiwei Pharmaceutical Co., Ltd.),

[0076] Glycerol (20191213, Zhejiang Suichang Huikang Pharmaceutical Co., Ltd.),

[0077] Sodium hydroxide (Pharmaceutical Excipients Registration Number: F20190001542 / A, Chengdu Huayi Pharmaceutical Excipients Manufacturing Co., Ltd.)

[0078] 2.2 Experimental procedure: Weigh 5.0 g of CTMB and 100.0 g of soybean oil for injection, place them in a beaker, heat to 70 - 80 °C under nitrogen protection, and stir to dissolve; then continue to weigh 12.0 g of egg yolk lecithin, add it to the above solution, and stir to dissolve to obtain the oil phase for standby. Weigh another 22.5 g of glycerol, measure about 680 mL of water, heat to 70 - 80 °C under nitrogen protection, and stir to dissolve to obtain the aqueous phase. Add the above oil phase to the aqueous phase, perform high-speed shearing for 5 - 15 minutes, adjust the pH to 8.5 - 11.0 with sodium hydroxide, and add water to make up to 1000 mL to prepare the primary emulsion. Then continue to homogenize the primary emulsion twice with a high-pressure homogenizer to make the average particle size of the homogenized emulsion droplets not more than 0.4 μm. The emulsion is filled into 5 mL glass ampoules under nitrogen protection and sterilized by heat pressing at 121 °C × 12 min to obtain TK-X07, in which the concentration of CTMB is 5 mg / mL. Prepare a blank emulsion injection without CTMB in the same method.

[0079] Example 3

[0080] Therapeutic effect of CTMB on acute ischemic stroke in rats induced by the suture occlusion method.

[0081] 3.1 Experimental materials: SPF grade SD rats (male, body weight 200 - 230 g, purchased from Sichuan Dashuo Experimental Animal Co., Ltd., certificate number: SCXK (Sichuan) 2020 - 0030),

[0082] MCAO suture (purchased from Beijing Xinuo Technology Co., Ltd., model: 2432 - A5)

[0083] The CTMB raw material drug was self-prepared in Example 1 (batch number: 20221107), and its emulsion injection was self-prepared in Example 2 (batch number: 20221114),

[0084] Edaravone dexborneol concentrated solution for injection (purchased from Simcere Pharmaceutical Co., Ltd. (specification: edaravone 10 mg / 5 mL, dexborneol 2.5 mg / mL; batch number: 180 - 220522)).

[0085] 3.2 Experimental grouping: Two hours after MCAO surgery, the Zea Longa score was used to determine whether the model was successful. Rats with successful modeling were randomly divided into groups and given drugs.

[0086] Rats were randomly divided into a sham-operated group (Sham group, given the same volume of blank emulsion as the high-dose TK-X07 group), a model group (Vehicle group, given the same volume of blank emulsion as the high-dose TK-X07 group), a low-dose TK-X07 group (CTMB-L group, 10 mg / kg), a high-dose TK-X07 group (CTMB-H group, 20 mg / kg), and an edaravone dexborneol injection concentrate group (EDB group, commercially available, 3 mg / kg), with 10 rats in each group. All rats were administered the drugs via intraperitoneal injection. (It should be noted that in the experiment, the rats were administered the drug via intraperitoneal injection. The effective dose ratio of the active ingredient CTMB in the emulsion injection to the human body mass was 0.2 mg to 4.0 mg / kg. This refers to the ratio of the effective dose of the active ingredient CTMB in the emulsion injection to the human body mass during intravenous infusion, and was calculated based on the effective dose in rats. Considering the differences in drug bioavailability, peak concentration, and time to peak concentration due to different species and administration methods, the effective dose ratio of the active ingredient CTMB in the emulsion injection to the human body mass was finally converted and determined to be 0.2 mg to 4.0 mg / kg.)

[0087] 3.3 Establishing an ischemia-reperfusion model using the suture occlusion method

[0088] Rats were fasted for 12 hours prior to surgery and anesthetized with 10% chloral hydrate (350 mg / kg, intraperitoneally). They were fixed in a supine position, and their body temperature was maintained at approximately 37°C. A midline incision was made in the neck, and the muscle and fascia were dissected along the inner edge of the sternocleidomastoid muscle. The right side was exposed, and the common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were bluntly dissected. Sutures were prepared at the proximal end of the CCA, ICA, and ECA. The proximal end of the CCA and ECA were ligated, and the ICA was temporarily clamped with an arterial clamp. A small hole was then made approximately 4 mm from the bifurcation of the CCA, and the suture was inserted into the ICA through the CCA. The arterial clamp on the ICA was released, and the suture was slowly advanced. When the marker on the suture reached the bifurcation and slight resistance was felt, the suture at the ICA was tightly secured. The wound was cleaned with saline and sutured. The sham surgery was performed identically to the experimental group except that no suture was inserted. After the rats regained consciousness from anesthesia, they were fed normally.

[0089] 3.4 Inclusion criteria for cerebral ischemia models

[0090] According to the Zea Longa neurological function score (Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke. 1989 Jan; 20(1):84-91. doi:10.1161 / 01.str.20.1.84.), the score was assessed 2 hours after the rats regained consciousness from anesthesia. Rats with a score of 1-2 were included in the group.

[0091] 0 points: No neurological deficits, normal activity level;

[0092] 1 point: Unable to fully extend the opposite forepaw;

[0093] 2 points: The animal was seen walking in circles;

[0094] 3 points: The body leans towards the side with hemiplegia;

[0095] 4 points: Unable to walk on their own, and has lost consciousness.

[0096] 3.5 Short-term neurological deficit score

[0097] Twenty-four hours after modeling, the neurological function of rats was comprehensively assessed using the modified mNSS score and Garcia score. The mNSS scoring criteria are shown in Table 1, and the Garcia scoring criteria are shown in Table 2. The rats' motor, sensory, climbing, and limb symmetry were evaluated. The mNSS score ranged from 0 to 18 points, with higher scores indicating more severe neurological impairment. The Garcia score ranged from 3 to 18 points, with lower scores indicating more severe neurological impairment. The scoring was completed independently by uninformed individuals who were not involved in modeling or drug administration.

[0098] Table 1. mNSS Neurological Function Scores

[0099]

[0100]

[0101] Table 2 Garcia Neurological Function Scores

[0102]

[0103]

[0104] As shown in Table 3, compared with the sham group, the model group (Vehicle group) showed a significantly higher mNSS score (P<0.01) and a significantly lower Garcia score (P<0.01) 24 hours after surgery, indicating significant neurological deficits in the model group rats 24 hours after MCAO surgery. Different doses of CTMB (CTMB-L and CTMB-H groups) and edaravone dexborneol injection concentrate (EDB group) all reduced mNSS scores and increased Garcia scores to varying degrees, improving the neurological deficits induced by MCAO. The CTMB-H group showed the most significant improvement (P<0.01), and its efficacy was superior to that of edaravone dexborneol, a drug clinically used to improve ischemic stroke. No toxic side effects related to CTMB administration were observed during the experiment.

[0105] Table 3 Short-term neurological deficit scores in rats

[0106]

[0107] Note: Compared with sham surgery (Sham group), ## P<0.01; compared with the model group (Vehicle group), **P<0.01, *P<0.05.

[0108] Example 4

[0109] Long-term therapeutic effect of CTMB on ischemic stroke induced by suture occlusion in rats.

[0110] 4.1 Neurological deficit score

[0111] Experimental materials, grouping (n=10-15), modeling method and scoring criteria were the same as for short-term treatment. Two hours after the suture occlusion method modeling, the drugs were administered according to the grouped dosing regimen. The drugs were then administered once a day for 14 days. The modified mNSS score was assessed on days 1, 4, 7 and 14.

[0112] As shown in Table 4, compared with the sham group, the model group (Vehicle group) had a significantly higher mNSS score on postoperative day 14 (P<0.01), indicating that the model group rats experienced significant neurological deficits 14 days after MCAO surgery. Both CTMB administration (CTMB-H group) and edaravone dexborneol injection concentrate group (EDB group) on days 1-7 reduced mNSS scores to varying degrees, improving the neurological deficits induced by MCAO. The CTMB-H group showed the most significant improvement, and its efficacy was slightly better than that of edaravone dexborneol injection concentrate, a drug clinically used to improve ischemic stroke. Furthermore, the CTMB-H group still significantly reduced mNSS scores on day 14. No toxic side effects related to CTMB administration were observed during the experiment.

[0113] Table 4 Long-term neurological deficit scores in rats

[0114]

[0115] Note: Compared with the sham surgery group (Sham group), ##P<0.01; compared with the model group (Vehicle group),

[0116] **P<0.01, *P<0.05.

[0117] 4.2 Adhesion Experiment

[0118] Before modeling, rats were trained for one week using an adhesion test, and rats that could successfully tear off the tape within 10 seconds were selected for modeling. Two hours after modeling using the suture method, the rats were immediately given the drug according to the grouped dosing regimen, and then continued to be given the drug once a day for 14 days. Adhesion tests were performed on days 1, 4, 7, 10, and 14 to assess the sensory and motor nerve function of the rats. The specific procedure was as follows: A circular tape with a diameter of approximately 12 mm was attached to the sole of the rat's left forelimb, and the time it took for the rat to sense and remove the tape was recorded. If the rat could not sense and / or remove the tape within 60 seconds, it was recorded as 60 seconds.

[0119] As shown in Table 5, compared with the sham group, the model group (Vehicle group) had a significantly longer sensory time on the adhesive tape 14 days after surgery (P<0.05), indicating that the sensory ability of the left forelimb of the rats in the model group was weakened 14 days after MCAO surgery; different doses of CTMB and EDB can promote the recovery of sensory ability in rats.

[0120] Table 5. Time for rats to perceive the tape

[0121]

[0122] Note: Compared with sham surgery (Sham group), ## P<0.01, # P<0.05; compared with the model group (Vehicle group),

[0123] **P<0.01, *P<0.05.

[0124] As can be seen from Table 6, compared with the sham operation group (Sham group), the time for the tape to fall off in the model group (Vehicle group) was significantly increased at 14 days after surgery (P<0.01), indicating that the motor ability of the left forelimb of the rats in the model group was weakened 14 days after MCAO; the CTMB-L group could significantly reduce the time for the rats to make the tape fall off from the 4th day (P<0.05), and the CTMB-H group could significantly reduce the time for the rats to make the tape fall off on the 10th day (P<0.05), and the drug effect was better than edaravone dexborneol, a drug clinically used to improve ischemic stroke. During the experiment, no toxic and side effects related to CTMB administration were observed.

[0125] Table 6 Time for rats to make the tape fall off

[0126]

[0127] Note: Compared with sham operation (Sham group), ## P<0.01, # P<0.05; compared with the model group (Vehicle group), **P<0.01, *P<0.05.

[0128] In summary, long-term administration of CTMB can significantly promote the recovery of motor function in rats and can restore the sensory ability of rats to a certain extent.

[0129] Example 5

[0130] Therapeutic effect of long-term administration of CTMB on a rat model of ischemic stroke induced by photochemical embolism.

[0131] 5.1 Experimental materials: SPF-grade SD rats. Male, weighing 200 - 230 g (purchased from Sichuan Dashuo Experimental Animal Co., Ltd., certificate number: SCXK(Sichuan)2020-0030)

[0132] Rose bengal (purchased from Sigma, USA)

[0133] The CTMB raw material drug was self-made in Example 1 (batch number: 20221107), and its emulsion injection was self-made in Example 2 (batch number: 20221114)

[0134] 5.2 Experimental grouping: After the rats were anesthetized and awake, a preliminary evaluation was carried out. The success criteria for model establishment were as follows: ① Flexion of the forelimb or hindlimb after model establishment ② Inability to walk in a straight line normally ③ The head deviated from the vertical axis by >10° within 30 s ④ Falling to the paretic side. The appearance of any one of the above 4 items indicated successful model establishment. The rats with successful model establishment were randomly grouped for drug administration.

[0135] Rats were randomly divided into a sham-operated group (Sham group, given the same volume of blank emulsion as the high-dose TK-X07 group), a model group (Vehicle group, given the same volume of blank emulsion as the high-dose TK-X07 group), a low-dose TK-X07 group (CTMB-L group, 10 mg / kg), and a high-dose TK-X07 group (CTMB-H group, 20 mg / kg), with 12 rats in each group. All rats were administered the medication via intraperitoneal injection.

[0136] 5.3 Establishment of an ischemic stroke model using photochemical embolization

[0137] Rats were fasted for 12 hours prior to surgery and anesthetized with 4% isoflurane. They were then fixed in a prone position using a stereotaxic apparatus, and anesthesia was maintained with 2% isoflurane. After disinfecting the skin of the head and neck with iodine, a longitudinal incision was made to expose the skull. The connective tissue membrane on the skull surface was peeled off. Using the Bregma point (0, 0, 0) as the origin, a 6mm diameter bone window was created centered 3.5mm to the right of the anterior fontanelle and 0.5mm below the anterior fontanelle (AP: 0.5mm, ML: 3.5mm). The dura mater was not damaged. Physiological saline was instilled until the blood vessels on the brain surface were clearly visible. Then, an 8mm diameter yellow-green laser beam was used for irradiation for 20 minutes. The sham surgery involved the same procedure as the experimental group, except that the rats did not undergo laser irradiation or receive rose blood injection. After recovery from anesthesia, the rats were fed normally.

[0138] 5.4 Neurological deficit score

[0139] The scoring criteria are the same as the mNSS scoring criteria under section 1.3. Two hours after photochemical embolization modeling, the drug was administered immediately according to the grouped dosing regimen, and then continued for 10 days, once a day. The modified mNSS score was performed on days 1, 3, 5, 7 and 10.

[0140] As shown in Table 7, compared with the sham group, the model group (Vehicle group) had a significantly higher mNSS score 10 days after surgery (P<0.01), indicating that the rats in the model group had significant neurological deficits 10 days after surgery. Different doses of CTMB administered from days 1 to 10 (CTMB-L and CTMB-H groups) all reduced the mNSS score to varying degrees and improved the neurological deficits induced by photochemical embolization, with the CTMB-L group showing the most significant improvement. No toxic side effects related to CTMB administration were observed during the experiment.

[0141] Table 7. Rats' mNSS neurological function scores

[0142]

[0143] Note: Compared with sham surgery (Sham group), ## P<0.01,# P<0.05; compared with the model group (Vehicle group), **P<0.01, *P<0.05.

[0144] 5.5 Adhesion Experiment

[0145] Before modeling, rats were trained for adhesion experiments for one week, and rats that could successfully tear off the tape within 10 seconds were selected for modeling. Two hours after modeling, the rats were immediately given the drug according to the grouped dosing regimen, and then continued to be given the drug once a day for 5 days. Adhesion experiments were performed on days 1, 3, and 5 to assess the sensory and motor nerve function of the rats. The specific procedure was as follows: A circular tape with a diameter of about 12 mm was attached to the sole of the left forelimb of the rat, and the time for the rat to sense and remove the tape was recorded. If the rat could not sense and / or remove the tape within 60 seconds, it was recorded as 60 seconds.

[0146] As shown in Table 8, compared with the sham group, the model group (Vehicle group) had a significantly longer sensory time on the adhesive tape 5 days after surgery (P<0.01), indicating that the sensory ability of the left forelimb of the rats in the model group was weakened 5 days after MCAO surgery; low-dose CTMB can significantly promote the recovery of sensory ability in rats from day 3.

[0147] Table 8. Time for rats to perceive the tape

[0148]

[0149] Note: Compared with sham surgery (Sham group), ## P<0.01, # P<0.05; compared with the model group (Vehicle group),

[0150] **P<0.01, *P<0.05.

[0151] As shown in Table 9, compared with the sham group, the model group (Vehicle group) had a significantly longer tape fall-off time 5 days after surgery (P<0.01), indicating that the left forelimb motor ability of rats in the model group was weakened 5 days after MCAO surgery; low-dose CTMB can significantly promote the recovery of rat motor ability from day 3.

[0152] Table 9. Time it took for the tape to fall off in rats.

[0153]

[0154] Note: Compared with sham surgery (Sham group), ## P<0.01, # P<0.05; compared with the model group (Vehicle group),

[0155] **P<0.01, *P<0.05.

[0156] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of CTMB in the preparation of drugs for the treatment and / or prevention of ischemic stroke, characterized in that, The CTMB structure is shown in Equation 1: Formula 1.

2. The application of CTMB according to claim 1 in the preparation of drugs for the treatment and / or prevention of ischemic stroke, characterized in that, The routes of administration for the drug include injection, oral administration, transdermal administration, inhalation, mucosal administration, or subcutaneous implantation.

3. The use of CTMB according to claim 1 or 2 in the preparation of drugs for the treatment and / or prevention of ischemic stroke, characterized in that, The drug is an injectable form.

4. The application of CTMB according to claim 3 in the preparation of drugs for the treatment and / or prevention of ischemic stroke, characterized in that, The injection is an emulsion injection.

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