Use of CTMB in preparation of drugs for treating and / or preventing traumatic brain injury

Abstract

The application provides application of CTMB in preparation of a drug for treating and / or preventing a traumatic brain injury disease, a milk injection for treating and / or preventing the TBI disease and a preparation method of the milk injection, and provides an effective, safe and non-toxic drug which can significantly improve the integrity of a blood brain barrier, promote recovery of motor function of a TBI model rat, reduce a neuroinflammatory response, antagonize glutamic excitatory neurotoxicity, reduce neuron apoptosis, improve nerve function defect and emotional anxiety, and has a significant neuroprotective effect on the TBI injury.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the use of CTMB in the preparation of drugs for the treatment and / or prevention of traumatic brain injury. Background Technology

[0002] Traumatic brain injury (TBI) is classified into primary and secondary injuries. Primary injury refers to direct damage to the brain caused by external force, occurring at the time of injury. Secondary injury refers to brain damage caused by cerebral ischemia and hypoxia, metabolic disorders, intracranial hematoma, increased intracranial pressure, etc. Traumatic brain injury (TBI) refers to brain injury caused by external force, mainly including open and closed injuries. It has a high incidence, high disability rate, and high mortality rate, and treatment costs are expensive. Patients often suffer from varying degrees of neurological function impairment. Currently, the severity of TBI, mainly caused by traffic accidents, is constantly increasing. Traffic accidents account for 13% of hospitalized TBI cases, but the mortality rate is as high as 58%. It is estimated that approximately 50 million people worldwide suffer from TBI each year, placing a huge economic burden on families and society.

[0003] In the short term following total brain injury (TBI), ischemia and hypoxia in the lesion area lead to energy depletion, resulting in a rapid and massive release of excitatory amino acids (glutamate and aspartate), while their reuptake is inhibited. This causes amino acid accumulation in the central nervous system, overactivating multiple downstream signaling pathways, leading to calcium influx and intracellular calcium overload, causing excitotoxic damage, progressive cell death, and permanent focal brain injury. Currently, routine resuscitation methods for TBI patients in clinical practice, such as endotracheal intubation, fluid resuscitation, and surgical treatment, are commonly used. Although there is a certain success rate in resuscitation, the results are still not ideal.

[0004] Clinically, drugs used to treat TBI mainly focus on treatment methods that can affect various damaging factors, among which the use of neuroprotective agents has received much attention. Their therapeutic effects mainly include: (1) Antibiotic treatment: Inhibition of autophagy leads to neurodegeneration, while the activation of microglia and astrocytes in the neuroimmune response causes the release of reactive oxygen species and cytokines. Therefore, enhancing anti-inflammatory and autophagy effects can improve the prognosis of TBI. Representative drugs include rapamycin and minocycline. (2) GABA A Receptor agonist: GABA A The receptor is activated by the endogenous neurotransmitter GABA, producing inhibitory Cl... - Current, thus enhancing GABA A The activity of receptors can reduce the membrane potential of TBI neurons and decrease neuronal excitability; a representative drug is benzodiazepine. (3) Cholinesterase inhibitors: Acetylcholine is an important neurotransmitter in the central cholinergic nervous system, participating in learning and memory physiological activities. Cholinesterase rapidly hydrolyzes acetylcholine at cholinergic synapses. Post-traumatic choline deficiency is a common cause of post-traumatic cognitive dysfunction. Therefore, cholinesterase inhibitors can improve cognitive function after TBI. Donepezil is a representative drug. (4) Ionoglutamate receptor inhibitors: Glutamate is an important neurotransmitter in the central nervous system. Ionoglutamate receptors are coupled with cation channels and are divided into three types: NMDA, KA, and AMPA. Glutamate-mediated neuronal hyperexcitability after TBI plays a key role in secondary neurological damage. Therefore, inhibitors of ionoglutamate receptors can treat excitotoxic neurological damage caused by TBI. Representative drugs include the NMDA receptor antagonist ketamine and the AMPA receptor antagonist perampanel. (5) Calcium channel blockers: Calcium ions enter cells through calcium ion channels, mediating nerve excitation. Calcium ion band blocking agents can bind to channel proteins and reduce intracellular calcium ion concentration. Therefore, calcium ion channel blockers can downregulate the excessive excitatory toxicity of nerve cells after TBI. Representative drugs are nifedipine and verapamil. (6) Sodium ion channel inhibitors: Voltage-gated sodium ion channels are the main ion channels constituting the rapid depolarization branch of neuronal action potentials. Therefore, inhibiting sodium ion channel currents can inhibit the generation and transmission of excessive action potentials after TBI. Representative drugs are carbamazepine and phenytoin sodium.

[0005] Currently, studies have found that neuroprotective agents have a protective effect against TBI in animal experiments and preclinical studies, but the efficacy in clinical trials is not ideal (Xiao Guomin. Research progress on the treatment of traumatic brain injury with pleiotropic neuroprotective drugs [J]. Health Research, 2011, 31(01):54-57.). Therefore, exploring new neuroprotective agents for the treatment of TBI is of great significance. Summary of the Invention

[0006] In summary, in order to address the problem that existing neuroprotective agents are not effective in treating traumatic brain injury in clinical practice, this invention provides the application of CTMB in the preparation of drugs for the treatment and / or prevention of traumatic brain injury.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides the use of CTMB in the preparation of drugs for the treatment and / or prevention of traumatic brain injury, wherein the structural formula of CTMB is shown in Formula I:

[0009]

[0010] Preferably, the traumatic brain injury disease includes secondary epilepsy, neurological or motor dysfunction caused by traumatic brain injury, and the route of administration of the drug includes injection, oral, transdermal, inhalation, mucosal administration or subcutaneous implantation.

[0011] Preferably, the drug is an injectable preparation.

[0012] Preferably, the injectable is an emulsion injection.

[0013] 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;

[0014] The effective dosage of the active ingredient CTMB in the emulsion injection, in ratio to rat unit mass, is 20 mg to 40 mg / kg.

[0015] 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.

[0016] Preferably, the emulsion injection further contains 0% to 2.5% glycerol.

[0017] Preferably, the oil phase is selected from one or more of soybean oil, medium-chain triglycerides, fish oil, olive oil, and structured triglycerides;

[0018] 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).

[0019] This invention also provides a method for preparing an emulsion injection, comprising the following steps:

[0020] (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.

[0021] (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.

[0022] (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.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The drug prepared by CTMB can significantly improve short-term neurological behavioral function, long-term motor function and restore post-traumatic anxiety in rats with traumatic brain injury;

[0025] (2) Improves the integrity of the blood-brain barrier and antagonizes the excitotoxicity of glutamate;

[0026] (3) It reduces cell apoptosis caused by mechanical damage and plays a neuroprotective role. CTMB is effective and safe, with no obvious toxic side effects.

[0027] By adopting the above technical solution, the present invention has the following beneficial effects: Experiments have confirmed that CTMB can improve the integrity of the blood-brain barrier, promote the recovery of motor function in model rats, reduce neuroinflammatory response, antagonize glutamate excitatory neurotoxicity, reduce neuronal apoptosis, improve neurological deficits and emotional anxiety in model rats, and has a significant neuroprotective effect on traumatic brain injury. Attached Figure Description

[0028] Figure 1 This is an evaluation of the rats' ability to adapt to new environments and their emotional anxiety.

[0029] Figure 2 The results of blood-brain barrier permeability testing for Experiment Example 3 (Evans blue dye content per gram of brain tissue in different groups of rats).

[0030] Figure 3 The results of detecting TNF-α, IL-1, and IL-6 in the cerebral cortex of rats in Experiment Example 3 are shown.

[0031] Figure 4 The results of GLT-1, GABA, and GLT-1 / GABA detection in different drug administration groups in Experiment Example 3 are shown.

[0032] Figure 5 Example 4 illustrates the effect of different concentrations of CTMB on the proliferation of HT22 cells damaged by mechanical scratches. Detailed Implementation

[0033] This invention provides the use of CTMB in the preparation of drugs for the treatment and / or prevention of traumatic brain injury, wherein the structural formula of CTMB is shown in Formula I:

[0034]

[0035] In this invention, the routes of administration of the drug include injection, oral administration, transdermal administration, inhalation, mucosal administration, or subcutaneous implantation.

[0036] In this invention, the drug is preferably an injection, and more preferably an emulsion injection.

[0037] The present invention also provides an emulsion injection for treating traumatic brain injury, 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.

[0038] In this invention, the emulsion injection solution comprises 0.5% to 5% CTMB, preferably 0.5% to 2%, more preferably 1%;

[0039] 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;

[0040] 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);

[0041] In this invention, the pH adjuster is a pharmaceutically acceptable base, selected from one or more of sodium hydroxide, sodium carbonate, and sodium bicarbonate;

[0042] In this invention, the emulsion injection solution includes water, preferably water for injection;

[0043] 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.

[0044] In this invention, the effective dosage of CTMB in the emulsion injection relative to human body mass is 0.2 mg to 4.0 mg / kg; the effective dosage of CTMB in the emulsion injection relative to rat body mass is 20 mg to 40 mg / kg. This ratio of 20 mg to 40 mg / kg is derived experimentally. In the experiment, rats were administered the drug via intraperitoneal injection. The ratio of 0.2 mg to 4.0 mg / kg refers to the ratio of the effective dosage of CTMB in the emulsion injection relative to human body mass during intravenous infusion, calculated from the effective dosage in rats. Considering differences in bioavailability, peak concentration, and time to peak concentration due to different species and administration methods, the ratio of the effective dosage of CTMB in the emulsion injection relative to human body mass was ultimately determined to be 0.2 mg to 4.0 mg / kg.

[0045] This invention also provides a method for preparing an emulsion injection, comprising the following steps:

[0046] (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.

[0047] (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.

[0048] (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.

[0049] In this invention, in step (1), CTMB is dissolved in an oil phase preheated to 70-80°C, preferably 78°C;

[0050] 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.

[0051] In this invention, in step (1), the preferred method of dissolving is to dissolve by stirring;

[0052] In this invention, the high-speed shearing time in step (2) is preferably 5 to 15 minutes, more preferably 10 minutes;

[0053] In this invention, the high-speed shearing speed in step (2) is preferably 13000 rpm;

[0054] In this invention, the pH value is preferably adjusted using sodium hydroxide in step (2);

[0055] In this invention, the pH value is preferably adjusted to 8.5 to 11.0 in step (2), and more preferably to 10.5;

[0056] In this invention, the high-pressure homogenization pressure in step (3) is preferably 800 bar;

[0057] In this invention, in step (3), the colostrum is homogenized under high pressure 1 to 3 times, preferably 2 times;

[0058] In this invention, the filtration in step (3) is preferably performed using a filter with a pore size of 5 μm;

[0059] In this invention, the sterilization in step (3) is preferably rotary autoclaving (121℃×12min, 103.4kPa).

[0060] 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.

[0061] In this invention, the CTMB has the following pharmacological effects:

[0062] (1) Improves the integrity of the blood-brain barrier and promotes the recovery of motor function in model rats;

[0063] (2) Regulate the level of inflammatory factors and reduce neuroinflammatory response;

[0064] (3) It antagonizes the excitatory neurotoxicity caused by excessive glutamate;

[0065] (4) Reduce the ratio of glutamate transporter 1 (GLT-1) / γ-aminobutyric acid (GABA) and restore the balance of excitatory amino acids (EAA) and inhibitory amino acids (IAA) in the brain;

[0066] (5) Improves neurological deficits and emotional anxiety in model rats.

[0067] In this invention, the drug can be used to: improve neurological or motor function damage (e.g., neurological or motor function damage caused by TBI), reduce early secondary brain injury (e.g., acute blood-brain barrier dysfunction caused by TBI), reduce acute mortality caused by TBI, prolong survival, improve emotional anxiety caused by TBI, and reduce brain tissue atrophy caused by TBI.

[0068] 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.

[0069] Example 1

[0070] Preparation of CTMB

[0071] 1.1 Raw materials used:

[0072] Cyclobutylcarboxylic acid (C13062789, Shanghai Maclean Biochemical Technology Co., Ltd.)

[0073] Oxaloyl chloride (JHSOWRS, Shanghai Xianding Biotechnology Co., Ltd.)

[0074] Dichloromethane (OPRN3RFE, Anhui Zesheng Technology Co., Ltd.)

[0075] 2,4,5-Trimethoxybenzaldehyde (T819646, Shanghai Maclean Biochemical Technology Co., Ltd.)

[0076] Aluminum trichloride (YZE8RSRV, Saas Chemical Technology (Shanghai) Co., Ltd.)

[0077] Sodium borohydride (2018041701, Chengdu Kelong Chemical Reagent Factory)

[0078] Tetrahydrofuran (T818767, Shanghai Maclean Biochemical Technology Co., Ltd.)

[0079] Acetic anhydride (2021123101, Chengdu Kelong Chemical Co., Ltd.)

[0080] Anhydrous copper sulfate (C10843174, Shanghai Maclean Biochemical Technology Co., Ltd.)

[0081] Anhydrous sodium acetate (Tianjin Kemio Chemical Reagent Co., Ltd.)

[0082] Anhydrous magnesium sulfate (Q / 12KM3936-2019, Tianjin Kemei Chemical Reagent Co., Ltd.), silica gel plate (10052521046809, Qingdao Haiyang Chemical Co., Ltd.).

[0083] 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%.

[0084] 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).

[0085] 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.

[0086] Example 2

[0087] Preparation of CTMB emulsion injection (abbreviated as TK-X07)

[0088] 2.1 Experimental Materials:

[0089] CTMB [1-(cyclobutyrylmethyl)-2,4,5-trimethoxybenzene] (prepared in Example 1),

[0090] Soybean oil for injection (DD20200603, Shandong Ruisheng Pharmaceutical Excipients Co., Ltd.)

[0091] Egg yolk lecithin (202008013, Shanghai Taiwei Pharmaceutical Co., Ltd.)

[0092] Glycerol (20191213, Zhejiang Suichang Huikang Pharmaceutical Co., Ltd.)

[0093] Sodium hydroxide (Pharmaceutical Excipient Registration Number: F20190001542 / A, Chengdu Huayi Pharmaceutical Excipients Manufacturing Co., Ltd.)

[0094] 2.2 Experimental procedure: Weigh 10.0 g of CTMB and 200.0 g of soybean oil for injection, place them in a beaker, heat to 70 - 80 °C under nitrogen protection, and stir to dissolve. Continuously weigh 12.0 g of egg yolk lecithin, add it thereto, and stir to dissolve to obtain the oil phase for standby. Separately weigh 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-mentioned oil phase to the aqueous phase, perform high-speed shearing for 5 - 15 minutes, add water to make up to 1000 mL, and prepare the primary emulsion. Continuously 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, adjust the pH to 8.5 - 11.0, fill the emulsion into 5 mL glass ampoules under nitrogen protection, and perform rotary heat pressing sterilization at 121 °C for 12 minutes to obtain TK-X07, in which the concentration of CTMB is 10 mg / mL. Prepare the blank emulsion injection without CTMB in the same method.

[0095] Experimental Example 1

[0096] Therapeutic effect of CTMB on acute TBI rat model

[0097] 1.1 Experimental materials:

[0098] SPF-grade SD rats, all male rats, weighing 240 - 260 g, purchased from Chengdu Dashuo Experimental Animal Co., Ltd., Sichuan Province, certificate number: SCXK (Sichuan) 2020 - 030.

[0099] The CTMB raw material drug was prepared according to the method of Example 1.

[0100] TK-X07 was prepared according to the method of Example 2.

[0101] Edaravone dexborneol injection concentrate was purchased from Simcere Pharmaceutical Co., Ltd. (specification: 10 mg: 2 mL; batch number: 1811283).

[0102] 1.2 Establishment of TBI rat model by mechanical impact method

[0103] Rats were fasted for 12 hours prior to surgery. Anesthesia was induced with 4% isoflurane and maintained with 2% isoflurane. The rats were immobilized in a supine position, and their body temperature was maintained at approximately 37°C. The rats were then fixed in a prone position on a stereotaxic apparatus. Hair on the top of the head was shaved, and routine disinfection was performed. A 1.0 cm incision was made in the midline of the skull, approximately 1.5 mm anterior to the lambdoid suture and 2.0 mm to the right of the midline, creating a 5.0 mm diameter circular bone window. Guided by the stereotaxic apparatus, a 40 g weight was dropped from a height of 15 cm in free fall, striking a 3 nm diameter impingement pin on the bone window in the brain (the exposed length of the impingement pin was approximately 3-4 nm), resulting in a circular hole approximately 3 mm in diameter in the rat's skull. One minute after the impact, the impingement pin was removed from the wound, and the wound was hemostatically controlled and sutured. After the surgery, the rats were placed in appropriate cages with controlled temperature (24±0.5℃). After they regained consciousness from anesthesia, they were fed normally.

[0104] 1.3 Inclusion criteria for the TBI rat model

[0105] Two hours after the rats regained consciousness following surgery, the model rats were scored according to the mNSS neurological function score. Rats with a score of 8-13 were selected for the next stage of the experiment. The mNSS neurological function scoring criteria are shown in Table 1.

[0106] Table 1. mNSS Neurological Function Scores

[0107]

[0108]

[0109] 1.4 Experimental Grouping

[0110] One hour after the mechanical impact, the mNSS score was used to determine whether the model was successful. Rats with successful modeling were used for the next step of the experiment.

[0111] Rats that successfully developed the model were randomly divided into 6 groups of 12 rats each:

[0112] Sham surgery group (P group): Administered the same volume of normal saline as the high-dose TK-X07 group;

[0113] Model group (T group): Administered the same volume of blank emulsion as the high-dose emulsion injection group;

[0114] Low-dose group (L group): TK-X07 was administered at a CTMB dose of 20 mg / kg;

[0115] Medium-dose group (M group): TK-X07 was administered at a CTMB dose of 30 mg / kg;

[0116] High-dose group (H group): TK-X07 was administered at a CTMB dose of 40 mg / kg;

[0117] Positive control group (Y group): Administered edaravone dexborneol concentrated solution for injection at a dose of 2 mg / kg;

[0118] 1.5 Neurological Function Scores

[0119] Two hours after the mechanical impact, all six groups received intraperitoneal injections once daily. Seven days after administration, the mNSS score was used to comprehensively assess the neurological function of rats in each group, evaluating motor function, sensation, climbing ability, and limb symmetry. The score ranged from 0 to 18 points, with higher scores indicating more severe neurological damage. The scoring was completed independently by uninformed individuals who were not involved in model establishment or drug administration. The scoring results are shown in Table 2.

[0120] Table 2 Short-term neurological deficit scores in rats

[0121]

[0122] Note: Compared with sham surgery (Group P), ### P<0.001; Compared with the model group (T group), ***P<0.01, **P<0.01, *P<0.05; Compared with the positive control (Y group), & P<0.05.

[0123] As shown in Table 2, compared with the sham surgery group (P group), the model group (T group) had a significantly higher mNSS score 7 days after TBI. ### P<0.001, indicating that the TBI rat model had significant neurological deficits; different doses of CTMB (L, M, H groups) and the positive control group (Y group) all significantly reduced mNSS scores and improved TBI-induced neurological deficits. Among them, the efficacy of the L, M, and H groups was better than that of the Y group, and the efficacy of the M group was significantly better than that of the Y group. & P<0.05).

[0124] Experimental Example 2

[0125] Neuroprotective effect of CTMB in a rat model of TBI

[0126] 2.1 Evaluation of the incidence of secondary epilepsy

[0127] The experimental materials and modeling methods were the same as in Example 1, with the grouping as follows:

[0128] Sham surgery group (P group): Received the same volume of normal saline as the CTMB group;

[0129] Model group (T group): Administered the same volume of blank emulsion as the CTMB group;

[0130] Low-dose group (L group): TK-X07 was administered at a CTMB dose of 20 mg / kg;

[0131] Medium-dose group (M group): TK-X07 was administered at a CTMB dose of 30 mg / kg;

[0132] High-dose group (H group): TK-X07 was administered at a CTMB dose of 40 mg / kg;

[0133] Positive control group (Group Y): Administered concentrated edaravone dexborneol injection solution at a dose of 2 mg / kg.

[0134] Two hours after TBI modeling, rats were administered drugs via intraperitoneal injection according to the respective drug administration regimens. The incidence of secondary epilepsy within 48 hours was observed, and the results are shown in Table 3.

[0135] Table 3. Incidence of acute epilepsy in TBI rats

[0136]

[0137] As shown in Table 3, the incidence of epilepsy in the T group rats within 48 hours was 41.6%, while the incidence of epilepsy in the L, M, H and Y groups was reduced, indicating that CTMB can reduce the rate of secondary epilepsy in TBI rats within 48 hours.

[0138] 2.2 Survival rate assessment

[0139] The experimental materials and modeling methods were the same as in Example 1, with the grouping as follows:

[0140] Sham surgery group (P group): Received the same volume of normal saline as the CTMB group;

[0141] Model group (T group): Administered the same volume of blank emulsion as the CTMB group;

[0142] Low-dose group (L group): TK-X07 was administered at a CTMB dose of 20 mg / kg;

[0143] Medium-dose group (M group): TK-X07 was administered at a CTMB dose of 30 mg / kg;

[0144] High-dose group (H group): TK-X07 was administered at a CTMB dose of 40 mg / kg;

[0145] Positive control group (Group Y): Administered concentrated edaravone dexborneol injection solution at a dose of 2 mg / kg.

[0146] Two hours after TBI modeling, rats were administered intraperitoneal injections according to their respective dosing regimens. The administration continued for 10 days, once a day. The survival rate of rats over 10 days was observed and recorded, and the results are shown in Table 4.

[0147] Table 4. Long-term survival rate of TBI rats

[0148]

[0149] As shown in Table 3, the survival rate of T group within 10 days was only 60%, while L, M, H and Y groups could improve the survival rate of TBI rats within 10 days.

[0150] 2.3 Assessment of adaptability to new environments and emotional anxiety

[0151] The open field test can be used to assess emotional anxiety in a rat model of short-term lithium injection (TBI). After the end of the survival observation and recording, the open field test was conducted on days 10 and 20 after TBI modeling, with the medium-dose group (M group) selected for the experiment. The open field was a square area with a length and width of 100 cm. After the start of the experiment, rats were released in the designated area according to the open field test guidelines (Smith DF. Biogenic amines and the effect of short-term lithium administration on open field activity in rats. Psychopharmacologia. 1975; 41(3):295-300. doi:10.1007 / BF00428940.PMID:125430.). The movement trajectory, movement distance, movement time, and other data of the rats in different areas were recorded using a computer tracking system (Noldus Ethovision, Tacoma, WA, USA).

[0152] Experimental results are as follows Figure 1 As shown, compared with group P, group T's movement distance was significantly reduced, while groups M and Y's movement distances were significantly higher than group T's; the time spent moving in the central region for each group was as follows: Figure 1 As shown on the right, compared with group P, group T showed a significant decrease in the number of shuttle movements and activity levels in the central region, while group M showed a significantly higher number of shuttle movements and activity levels in the central region than group T. In conclusion, CTMB administration can significantly improve the adaptability of TBI rats to new environments and alleviate emotional anxiety.

[0153] (Note: Compared with sham surgery (Group P),) # P<0.01; compared with the model group (T group), ***P<0.01, **P<0.01, *P<0.05).

[0154] Experimental Example 3

[0155] Research on the mechanism of action of CTMB in the treatment of traumatic brain injury

[0156] Experimental materials:

[0157] Evans Blue (C11891158, Shanghai Maclean Biotechnology);

[0158] Formamide (20190716, Tianjin Bodi Chemical);

[0159] IL-1 Detection Kit (E-EL-H0149c, Wuhan Yilairuit Biotechnology Co., Ltd.);

[0160] IL-6 Detection Kit (E-EL-H2518c, Wuhan Yilairuit Biotechnology Co., Ltd.);

[0161] TNF-α Detection Kit (9680019151122, Aibote Biotechnology);

[0162] GABA detection kit (ZD0342B47674, Wuhan Yilairuit Biotechnology Co., Ltd.);

[0163] GLT-1 Detection Kit (Apr 2023, Quanzhou Ruixin Biotechnology Co., Ltd.)

[0164] 3.1 Blood-brain barrier permeability detection

[0165] The experimental grouping and dosing regimens are as follows:

[0166] Sham surgery group (P group): Received the same volume of normal saline as the CTMB group;

[0167] Model group (T group): Administered the same volume of blank emulsion as the CTMB group;

[0168] Medium-dose group (M group): TK-X07 was administered at a dose of 30 mg / kg.

[0169] Two hours after TBI modeling, rats in each group were administered the drug via intraperitoneal injection. Twenty-four hours later, 4% Evans blue solution (2.5 mL / kg) was injected into the right tail vein of the rats. One hour later, the rats were deeply anesthetized, and 100 mL of physiological saline was perfused through the heart. The rats were then quickly decapitated, and the brains were immediately separated into the left hemisphere, right hemisphere, cerebellum, and brainstem. The wet weight of the brain tissue was measured, and the tissue was immersed in 10 times its volume of pure formamide, incubated at 60°C for 48 hours, centrifuged at 25°C and 10,000 rpm for 30 minutes, and the supernatant was collected. Evans blue dye was detected at 622 nm using ultraviolet spectrophotometry. A standard curve was plotted for quantification, and the final result was shown as the Evans blue content per gram of brain tissue (μg / g). Results are as follows: Figure 2As shown (n=6), compared with the sham surgery group (P group), the permeability of the blood-brain barrier in the hemorrhage side of the cerebral hemisphere in the model group (T group) increased 24 hours after surgery, while the medium dose of TK-X07 (M group) group significantly reduced Evans blue exudation and improved the integrity of the blood-brain barrier.

[0170] 3.2 Detection of IL-1, IL-6 and TNF-α

[0171] The experimental grouping and administration regimens were the same as in section 3.1. Two hours after TBI modeling, rats in different groups were administered the drug intraperitoneally. Twenty-four hours after administration, the rats were deeply anesthetized, decapitated, and the brain was harvested. Approximately 60–120 mg of the hematogenous cerebral cortex was extracted and added to 10 times the volume of ice-cold centrifugation buffer for biochemical analysis. The mixture was homogenized for 10 min on ice, centrifuged at 4°C and 14,000 rpm / min for 30 min, and the supernatant was collected to detect the levels of inflammatory factors IL-1, IL-6, and TNF-α according to the instructions of the IL-1, IL-6, and TNF-α assay kits. The results are as follows: Figure 3 As shown (n=5-8), compared with the P group, the levels of IL-1, IL-6 and TNF-α in the hemorrhagic cerebral hemisphere of the T group rats were significantly increased 24 hours after surgery, while the M group could significantly reduce the levels of pro-inflammatory cytokines TNF-α, inflammatory factors IL-1 and IL-6 in the cerebral cortex of TBI rats, which is beneficial to reduce neuroinflammation and improve the motor function of rats.

[0172] 3.3 Determination of GLT-1 and GABA content

[0173] The experimental grouping and administration regimens were the same as described in section 3.1. After 7 consecutive days of administration, rats were deeply anesthetized, decapitated, and their brains were harvested. Approximately 60–120 mg of the hematogenous cerebral cortex was extracted and added to 10 times its volume of ice-cold centrifugation buffer for biochemical analysis. The mixture was homogenized for 10 min on ice, centrifuged at 4°C and 14,000 rpm / min for 30 min, and the supernatant was collected to determine the GLT-1 and GABA levels according to the instructions of the GLT-1 and GABA content assay kits. Glutamate transporter 1 (GLT-1) is an important glutamate transporter in the brain, which transports extracellular glutamate into intracellular cells. Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter; under pathological conditions, a sharp increase in extracellular glutamate concentration can lead to neuronal overexcitogenic toxicity. The figure shows the GLT-1 and GABA levels and their ratio measured in different administration groups, which can be used to assess the excitogenic neurotoxicity caused by TBI injury. The results are as follows: Figure 4As shown (n=6), compared with the P group, the GLT-1 content and GLT-1 / GABA ratio of the hemorrhagic side of the T group rats were significantly increased 7 days after surgery. In contrast, the administration of the M group could reduce the GLT-1 content in the brain of TBI rats, increase the GABA content, significantly reduce the GLT-1 / GABA ratio, restore the balance of excitatory amino acids (EAA) and inhibitory amino acids (IAA) in the brain, reduce the excitatory neurotoxicity caused by excessive glutamate, and thus improve the motor function of rats.

[0174] Experiment Example 4

[0175] CTMB's protective effect against mechanically scratched neurons

[0176] 4.1 Experimental Materials:

[0177] The HT22 cell line was purchased from Wuhan Pronosai Life Science Technology Co., Ltd.

[0178] CCK8 (C12029690, Sigma-Aldrich, USA);

[0179] DMEM high glucose medium (AG29301810, Hyclone, USA);

[0180] Fetal bovine serum (20010401, Gibco, USA)

[0181] PBS powder (WK173618-1, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.);

[0182] DMSO (20201220, Beijing Solarbio Technology Co., Ltd.).

[0183] 4.2 Experimental Procedure

[0184] Preparation of complete culture medium: Mix DMEM high glucose medium, fetal bovine serum and penicillin-streptomycin solution (double antibiotic) at a volume ratio of 90:9:1 and store at 4℃.

[0185] Preparation of serum-free culture medium: Mix DMEM high glucose medium and penicillin-streptomycin solution (double antibiotic) at a volume ratio of 99:1 and store at 4℃.

[0186] HT22 cells were cultured in complete medium and, when in the logarithmic growth phase, were injected with 1×10⁻⁶ cells / mL. 4 Seed 100 μL / well of cells into 96-well plates, fill the edge wells with sterile PBS, and incubate at 37°C and 5% CO2 for 24 h until cells are fully adherent; discard the supernatant.

[0187] Drug treatment groups: 100 μL of CTMB diluted with serum-free medium at different concentrations (to final concentrations of 0.3 μM, 0.625 μM, 1.25 μM, and 2.5 μM, respectively) was added. After culturing for 2 h, three horizontal and three vertical lines were drawn into each of the above drug treatment groups to establish a mechanical scratch damage model. The groups were then cultured at 37℃ and 5% CO2 for another 24 h.

[0188] Control group: The same volume of serum-free culture medium was added for culture, and no scratch damage was performed. All other operations were the same as those in the drug treatment group.

[0189] Model group: The same volume of serum-free culture medium was added sequentially and mechanical scratch damage was performed before culture. The remaining operations were the same as those for the drug-treated group.

[0190] Subsequently, 10 μL of CCK was added to each well, and the mixture was incubated at 37°C and 5% CO2 for 40 min. The OD value at 570 nm was then measured using a microplate reader. The results are as follows: Figure 5 As shown.

[0191] like Figure 5 As shown, compared with the control group (cell proliferation activity was 1.0), the model group HT22 cells underwent apoptosis or necrosis due to mechanical scratch damage, and the proliferation activity was significantly reduced. On the other hand, CTMB at concentrations of 0.3 μM to 2.5 μM could significantly improve the decrease in cell proliferation activity caused by mechanical scratch damage, suggesting that it can alleviate cell apoptosis and necrosis caused by mechanical damage.

[0192] As can be seen from the above embodiments, the present invention provides the application of CTMB in the preparation of drugs for the treatment or prevention of traumatic brain injury.

[0193] 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 application of CTMB in the preparation of drugs for the treatment and / or prevention of traumatic brain injury, characterized in that, The structural formula of the CTMB is shown in Formula I: Equation I.

2. The application according to claim 1, characterized in that, The traumatic brain injury disease includes secondary epilepsy, neurological or motor dysfunction caused by traumatic brain injury, and the drug can be administered via injection, oral, transdermal, inhalation, mucosal administration or subcutaneous implantation.

3. The application according to claim 1, characterized in that, The drug is an injectable form.

4. The application according to claim 3, characterized in that, The injection is an emulsion injection.

5. The application according to claim 4, characterized in that, The effective dosage of the active ingredient CTMB in the emulsion injection, in ratio to human body mass, is 0.2 mg to 4.0 mg / kg. The effective dosage of the active ingredient CTMB in the emulsion injection is 20 mg to 40 mg / kg per unit weight of rat.

6. A creamy injection solution of CTMB for the treatment and / or prevention of traumatic brain injury, characterized in that, It includes 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; The structural formula of the CTMB is shown in Formula I: Equation I.

7. The emulsion injection solution according to claim 6, characterized in that, The emulsion injection also contains 0% to 2.5% glycerol.

8. The emulsion injection solution according to claim 6, characterized in that, The oil phase is selected from one or more of soybean oil, medium-chain triglycerides, fish oil, olive oil, and structured triglycerides; The emulsifier is selected from one or more of egg yolk lecithin, soybean lecithin, Pluronic F 68, and polyethylene glycol stearic acid-15.

9. The method for preparing the emulsion injection solution according to any one of claims 6 to 8, characterized in that, Includes the following steps: (1) Under nitrogen or inert gas protection, CTMB is dissolved in an oil phase preheated to 70~80℃, and then the emulsifier is dissolved in the oil phase in which CTMB is dissolved or in an aqueous phase at 70~80℃. (2) The oil phase and aqueous phase prepared above are mixed by high-speed shearing to prepare the primary emulsion, and the pH value is adjusted. (3) The colostrum is homogenized under high pressure 1 to 3 times until the average droplet size is ≤0.4 μm, filtered, and sterilized by rotary autoclaving to obtain an emulsion injection containing CTMB.

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