CN117695260A

CN117695260A - Application of CTMB in preparation of medicines for treating and/or preventing traumatic craniocerebral injury diseases

Info

Publication number: CN117695260A
Application number: CN202311776442.0A
Authority: CN
Inventors: 毛声俊; 严睿洁; 廖灿; 李蕊; 张检; 周青松
Original assignee: Chengdu Xinrui Taikang Technology Co ltd
Current assignee: Chengdu Xinrui Taikang Technology Co ltd
Priority date: 2023-12-21
Filing date: 2023-12-21
Publication date: 2024-03-15

Abstract

The invention provides an application of CTMB in preparing a medicament for treating and/or preventing traumatic craniocerebral injury diseases, an emulsion injection for treating and/or preventing TBI diseases and a preparation method thereof, and provides a medicament which can obviously improve blood brain barrier integrity, promote motor function recovery of a TBI model rat, alleviate neuroinflammatory reaction, antagonize glutamate excitatory neurotoxicity, reduce neuronal apoptosis, improve neurological deficit and emotional anxiety, has obvious neuroprotection effect on TBI injury, is safe, and has no obvious toxic or side effect.

Description

Application of CTMB in preparation of medicines for treating and/or preventing traumatic craniocerebral injury diseases

Technical Field

The invention relates to the technical field of medicines, in particular to application of CTMB in preparing medicines for treating and/or preventing traumatic craniocerebral injury diseases.

Background

Craniocerebral injury is classified as primary and secondary. The primary injury refers to direct injury caused by external force acting on the brain, and occurs at the time of injury; the secondary injury refers to brain injury caused by cerebral ischemia and anoxia, metabolic disorder, intracranial hematoma, intracranial pressure increase and the like. Traumatic craniocerebral injury (Traumatic brain injury, TBI) refers to brain injury caused by external force, mainly comprises open injury and closed injury, has high incidence rate, high disability rate and high mortality rate, is expensive to treat, and patients often leave nerve function injury with different degrees. At present, the severity of craniocerebral injury mainly caused by traffic accidents is increased continuously, and the craniocerebral injury caused by the craniocerebral injury occupies 13% of patients in hospital, but the death rate is as high as 58%. It is counted that approximately five tens of millions of people suffer from TBI each year worldwide, creating a huge economic burden for the home and society.

Short term after TBI occurs, ischemic and hypoxic trauma in the focal area causes energy exhaustion, excitatory amino acids (glutamate and aspartate) are released rapidly in large amounts, while their reuptake is inhibited, resulting in amino acid accumulation in the central system, excessive activation of multiple downstream signaling pathways, causing calcium influx and intracellular calcium overload, resulting in excitotoxic damage, resulting in progressive cell death and permanent focal brain damage. At present, conventional rescue is usually adopted for TBI patients in clinic, such as tracheal intubation, liquid resuscitation, operation treatment and the like, and although a certain rescue success rate exists, the effect is still not ideal.

Drugs for treating TBI clinically have focused mainly on therapeutic approaches that can affect various injury factors, with the use of neuroprotective agents being of great concern. The treatment effect mainly comprises: (1) antibiotic treatment: inhibition of autophagy results in neurodegeneration, whereas microglial and astrocyte activation in neuroimmune response causes active oxygen cluster, cytokine release, thus enhancing anti-inflammatory and autophagy can improve TBI prognosis damage, with rapamycin and meromycin being representative drugs. (2) GABA (gamma-amino-acid-gamma _A Receptor agonists: GABA (gamma-amino-acid-gamma _A The receptor is activated by the endogenous neurotransmitter GABA, producing inhibitory Cl ^- Current flow, thus enhancing GABA _A The activity of the receptor can reduce the potential of TBI nerve cell membrane, reduce the excitability of neurons, and represent the medicine benzodiazepineAnd (5) tosylate-like material Malun. (3) cholinesterase inhibitors: acetylcholine is an important transmitter of the central cholinergic nervous system, involved in learning and memory physiological activities, and cholinesterase rapidly hydrolyzes acetylcholine at cholinergic synapses. The posttraumatic choline deficiency is a common cause of posttraumatic cognitive dysfunction, so cholinesterase inhibitors can improve the posttbi cognitive function, representing the drug donepezil. (4) inhibitors of the ionotropic glutamate receptor: glutamate is an important neurotransmitter in the central nervous system, and ionotropic glutamate receptors are coupled to cation channels and are classified into three types, NMDA, KA and AMPA. Glutamate-mediated neuronal hyperexcitability after TBI occurrence plays a key role in secondary nerve injury, and thus inhibition of the ionotropic glutamate receptor inhibitors may treat excitotoxic nerve injury caused by TBI. Wherein the representative drugs are NMDA receptor antagonists ketamine and AMPA receptor antagonist pirenzenepamil. (5) calcium channel blockers: calcium ions enter cells through calcium ion channels to mediate nerve excitation, and calcium ion passband blocker can be used with channel proteinsThe combination reduces the intracellular calcium ion concentration, so that the calcium channel blocker can down regulate the hyperexcitability toxicity of nerve cells after TBI, and represents medicines nifedipine and verapamil. (6) sodium ion channel inhibitors: the voltage-gated sodium ion channel is a main ion channel forming a rapid depolarization branch of the action potential of the neuron, so that the inhibition of the current of the sodium ion channel can inhibit the generation and transmission of excessive action potential after TBI, and the representative drugs are carbamazepine and phenytoin sodium.

At present, the neuroprotectant has been found to have a protective effect on TBI in animal experiments and preclinical studies, but the clinical test has a poor curative effect (Shounational. Traumatic brain injury multi-effect neuroprotection drug treatment research progress [ J ]. Health study, 2011,31 (01): 54-57.). Thus, the search for new neuroprotective agents would be of great interest in the treatment of TBI.

Disclosure of Invention

In summary, in order to solve the problem that the neuroprotectant in the prior art has poor clinical curative effect on traumatic brain injury, the invention provides the application of CTMB in preparing medicaments for treating and/or preventing traumatic brain injury diseases.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides application of CTMB in preparation of medicines for treating and/or preventing traumatic craniocerebral injury diseases, wherein the structural formula of the CTMB is shown as formula I:

preferably, the traumatic brain injury disease comprises secondary epilepsy, neurological or motor dysfunction caused by traumatic brain injury, and the administration route of the drug comprises injection, oral administration, transdermal administration, inhalation, mucosal administration or subcutaneous implantation.

Preferably, the medicament is an injection.

Preferably, the injection is a emulsion injection.

Preferably, the ratio of the effective dosage of the effective component CTMB in the emulsion injection to the unit mass of human body is 0.2 mg-4.0 mg/kg;

the ratio of the effective dosage of the effective component CTMB in the emulsion injection to the unit mass of the rat is 20 mg-40 mg/kg.

The invention also provides an emulsion injection for treating ischemic cerebral apoplexy, which comprises the following components in percentage by weight: 0.5 to 5 percent of CTMB, 5 to 30 percent of oil phase, 0.6 to 1.8 percent of emulsifier, 0.001 to 0.01 percent of pH regulator and the balance of water.

Preferably, the emulsion injection further comprises 0% -2.5% of glycerol.

Preferably, the oil phase is selected from one or more of soybean oil, medium chain triglycerides, fish oil, olive oil and structural triglycerides;

the emulsifier is one or more selected from egg yolk lecithin, soybean lecithin, pluronic F68 and polyethylene glycol stearic acid-15 (Solutol HS 15).

The invention also provides a preparation method of the emulsion injection, which comprises the following steps:

(1) Under the protection of nitrogen or inert gas, dissolving CTMB in an oil phase preheated to 70-80 ℃, and then dissolving an emulsifier in the oil phase dissolving CTMB or in a water phase at 70-80 ℃;

(2) Mixing the oil phase and the water phase by high-speed shearing to prepare colostrum, and regulating the pH value;

(3) Homogenizing the colostrum under high pressure for 1-3 times until the average particle diameter of emulsion drops is less than or equal to 0.4 mu m, filtering, and performing rotary hot-press sterilization to obtain the emulsion injection containing CTMB.

Compared with the prior art, the invention has the following beneficial effects:

(1) The medicine prepared by using CTMB can obviously improve the short nerve behavioural function and the long-term motor function of a rat with traumatic brain injury and recover the anxiety of the rat after the injury;

(2) Improving blood brain barrier integrity, antagonizing glutamate excitotoxicity;

(3) Reduces apoptosis caused by mechanical injury, plays a role in neuroprotection, is effective and safe for CTMB, and has no obvious toxic and side effects.

By adopting the technical scheme, the invention has the following beneficial effects: experiments prove that CTMB can improve the integrity of blood brain barriers and promote the recovery of the motor functions of model rats; reducing the neuroinflammatory response; antagonizing glutamate excitotoxicity; reducing neuronal apoptosis; the nerve function defect and emotional anxiety of the model rat are improved, and the nerve protection effect on traumatic craniocerebral injury is obvious.

Drawings

FIG. 1 shows the evaluation results of the adaptability of rats to new environments and emotional anxiety.

FIG. 2 shows the results of the blood brain barrier permeability test of Experimental example 3 (Evansan dye content per gram of brain tissue of rats of different groups).

FIG. 3 shows the results of detection of TNF-alpha, IL-1 and IL-6 in rat cerebral cortex of experimental example 3.

FIG. 4 shows the results of GLT-1, GABA and GLT-1/GABA assays of different dosing groups of experimental example 3.

FIG. 5 shows the effect of CTMB at various concentrations on the proliferation activity of mechanical scratch-damaged HT22 cells of experimental example 4.

Detailed Description

in the present invention, the administration route of the drug includes injection, oral administration, transdermal administration, inhalation, mucosal administration or subcutaneous implantation.

In the present invention, the drug is preferably an injection, and more preferably an emulsion injection.

The invention also provides an emulsion injection for treating traumatic craniocerebral injury diseases, which comprises the following components in percentage by weight: 0.5 to 5 percent of CTMB, 5 to 30 percent of oil phase, 0.6 to 1.8 percent of emulsifier, 0.001 to 0.01 percent of pH regulator and the balance of water.

In the present invention, the emulsion injection comprises CTMB 0.5% to 5%, preferably 0.5% to 2%, more preferably 1%;

in the invention, the emulsion injection comprises 5% -30%, preferably 10% -20%, more preferably 10% of oil phase, wherein the oil phase is selected from one or more of soybean oil, medium chain triglyceride, fish oil, olive oil and structural triglyceride;

the emulsion injection in the invention comprises 0.6 to 1.8 percent of emulsifying agent, preferably 0.6 to 1.5 percent, more preferably 1.2 percent; the emulsifier is one or more selected from egg yolk lecithin, soybean lecithin, pluronic F68 and polyethylene glycol stearic acid-15 (Solutol HS 15);

in the invention, the pH regulator is pharmaceutically acceptable alkali, and is selected from one or more of sodium hydroxide, sodium carbonate and sodium bicarbonate;

in the present invention, the emulsion injection comprises water, preferably water for injection;

in the present invention, the emulsion injection preferably further comprises 0% to 2.5%, preferably 2.0% to 2.5%, further preferably 2.25% of glycerol, which acts as an osmotic pressure regulator;

in the invention, the ratio of the effective dosage of the effective component CTMB in the emulsion injection to the unit mass of human body is 0.2 mg-4.0 mg/kg; the ratio of the effective dosage of the effective component CTMB in the emulsion injection to the unit mass of the rat is 20 mg-40 mg/kg; in the present invention, the ratio of the effective amount of CTMB as an active ingredient in the emulsion injection to the unit mass of the rat is 20mg to 40mg/kg, which is obtained by experiments. In experiments, the rat is administrated by intraperitoneal injection, the ratio of the effective component CTMB in the emulsion injection to the unit mass of human body is 0.2-4.0 mg/kg, the ratio of the effective component CTMB in the emulsion injection to the unit mass of human body during intravenous drip is calculated by the effective component of the rat, and the ratio of the effective component CTMB in the emulsion injection to the unit mass of human body is finally determined to be 0.2-4.0 mg/kg in consideration of different drug bioavailability, different drug peak concentration and peak time caused by different administration modes.

In the present invention, CTMB is dissolved in the oil phase preheated to 70-80 ℃ in the step (1), preferably 78 ℃;

in the present invention, the step (1) is to dissolve the emulsifier in the oil phase for dissolving CTMB or in the water phase at 70-80 ℃, preferably at 78 ℃;

in the present invention, the dissolving in the step (1) is preferably stirring to dissolve;

in the present invention, the high-speed shearing time in the step (2) is preferably 5 to 15 minutes, more preferably 10 minutes;

in the present invention, the high-speed shearing rotation speed in the step (2) is preferably 13000rpm;

in the present invention, the pH value is preferably adjusted by sodium hydroxide in the step (2);

in the present invention, the pH value is preferably adjusted to 8.5 to 11.0, more preferably 10.5 in the step (2);

in the present invention, the high-pressure homogenizing pressure in the step (3) is preferably 800bar;

in the present invention, the colostrum is homogenized 1 to 3 times, preferably 2 times, under high pressure in step (3);

in the present invention, the filtration in the step (3) is preferably filtration with a filter having a retention pore size of 5. Mu.m;

in the present invention, the sterilization in the step (3) is preferably a rotary heat press sterilization (121 ℃ C..times.12 min,103.4 kPa).

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

In the invention, the CTMB has the following pharmacological actions:

(1) Improving the integrity of the blood brain barrier and promoting the recovery of the motor function of the model rat;

(2) Modulating inflammatory factor levels, reducing neuroinflammatory responses;

(3) Antagonizing excitotoxicity caused by excess glutamate;

(4) Lowering the glutamate transporter 1 (GLT-1)/gamma-aminobutyric acid (GABA) ratio, restoring the balance of Excitatory Amino Acids (EAA) and Inhibitory Amino Acids (IAA) in the brain;

(5) Improving neurological deficit and emotional anxiety in the model rats.

In the present invention, the drug may be used for: improving damage to nerve or motor function (e.g., damage to nerve or motor function due to TBI), alleviating early secondary brain damage (e.g., acute phase brain tissue blood brain barrier dysfunction due to TBI), reducing acute phase mortality due to TBI, prolonging survival, improving emotional anxiety due to TBI, and alleviating brain tissue atrophy due to TBI.

Example 1

Preparation of CTMB

1.1 raw materials used:

cyclobutyl formic acid (C13062789, shanghai Michlin Biochemical Co., ltd.),

Oxalyl chloride (JHSOWRS, shanghai Jiding Biotechnology Co., ltd.),

Methylene chloride (OPRN 3RFE, anhui Zernike science, inc.),

2,4, 5-trimethoxybenzaldehyde (T819646, shanghai Michelin Biochemical Co., ltd.),

Aluminum trichloride (YZE RSRV, satsu chemical Co., ltd.),

Sodium borohydride (2018041701, chengdu Kelong chemical reagent Co., ltd.),

Tetrahydrofuran (T818767, shanghai Michlin Biochemical technologies Co., ltd.),

Acetic anhydride (2021123101, chengdu chemical Co., ltd.),

Anhydrous copper sulfate (C10843174, shanghai Michlin Biochemical technologies Co., ltd.),

Anhydrous sodium acetate (Tianjin chemical agent Co., ltd.),

Anhydrous magnesium sulfate (Q/12 KM3936-2019, MIEuro chemical Co., tianjin, inc.), silica gel plate (10052521046809, qingdao ocean chemical Co., ltd.).

1.2 preparation process: taking cyclobutyl formic acid in a three-necked flask in N ₂ Dissolved in 150mL of anhydrous dichloromethane under protection, and stirred at normal temperature. Oxalyl chloride was gradually added dropwise, stirred at room temperature until no bubbles were generated, and concentrated to give 25.0g of an orange-yellow intermediate liquid. The liquid obtained above was dissolved in 100mL of anhydrous methylene chloride, aluminum trichloride was added at 0℃and the temperature was raised to room temperature, stirred for 2.5 hours, then 300mL of water was added for quenching, extraction was performed with ethyl acetate (3X 200 mL), the organic phases were combined, dried over anhydrous magnesium sulfate and concentrated to give a crude product, which was slurried with 40mL of ethanol for 1 hour and purified to give 43.7g of a white solid. The solid prepared above was dissolved in 150mL of tetrahydrofuran, 20mL of aqueous sodium borohydride solution and 10 drops of 10% sodium hydroxide solution were added dropwise at 0 ℃ and heated to 60 ℃, stirred for 3 hours under heating, cooled to 37 ℃ and then adjusted to pH 7 to 8 with 1N hydrochloric acid solution, tetrahydrofuran was removed by concentration, extracted with ethyl acetate (3×200 mL), the organic phases were combined, dried over anhydrous magnesium sulfate and concentrated to give 42.6g of orange-yellow semi-solid (compound 1 f), anhydrous sodium acetate (8.31 g,101.25 mmol) was added to 210mL of acetic anhydride and heated to 140 ℃, heated for 3 hours, acetic anhydride was removed by concentration, 200mL of water was added, extracted with ethyl acetate (4×200 mL), the organic phases were combined, dried over anhydrous magnesium sulfate and concentrated to give a crude product, which was recrystallized and purified with 70% ethanol to give 23.47g of CTMB as a white solid, yield: 59.4%.

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

13C-NMR(100MHz，CDCl3)δ150.7，148.0，143.1，142.5，119.0，114.5，111.4，98.0，56.9，56.6，56.2，32.8，32.6，18.5。

Example 2

Preparation of CTMB emulsion injection (TK-X07 for short)

2.1 experimental materials:

CTMB [1- (Cyclobutylidenemethyl) -2,4, 5-trimethoxybenzene ] (example 1 self-made)

Soybean oil for injection (DD 20200603, shandong Rui crude drug auxiliary material Co., ltd.),

Egg yolk lecithin (202008013, shanghai Taiwei pharmaceutical Co., ltd.),

Glycerol (20191213, zhejiang Seischikang pharmaceutical Co., ltd.),

Sodium hydroxide (pharmaceutic adjuvant registration number F20190001542/A, chengdu Hua Yi pharmaceutic adjuvant manufacturing Co., ltd.)

2.2 experimental procedure: weighing 10.0g of CTMB and 200.0g of soybean oil for injection, placing the materials into a beaker, heating to 70-80 ℃ under the protection of nitrogen, and stirring to dissolve; weighing 12.0g of egg yolk lecithin, adding the egg yolk lecithin into the egg yolk lecithin, stirring the egg yolk lecithin to dissolve the egg yolk lecithin, and preparing an oil phase for later use. Weighing 22.5g of glycerol, weighing 680mL of water, heating to 70-80 ℃ under the protection of nitrogen, and stirring to dissolve; an aqueous phase is obtained. Adding the oil phase into the water phase, shearing at high speed for 5-15 minutes, and adding water to 1000mL to prepare the colostrum. Homogenizing the primary emulsion for 2 times by a high-pressure homogenizer to ensure that the average particle diameter of the homogenized emulsion drops is not more than 0.4 mu m, regulating the pH value to 8.5-11.0, filling the emulsion into a 5mL glass ampoule under the protection of nitrogen filling, and performing rotary hot-press sterilization at 121 ℃ for 12min to obtain TK-X07, wherein the concentration of CTMB is 10mg/mL. A blank emulsion injection without CTMB was prepared in the same manner.

Experimental example 1

Therapeutic effects of CTMB on acute-phase TBI rat model

1.1 experimental materials:

SPF SD rats, all male rats, weighing 240-260 g, purchased from Mida laboratory animal Limited, sichuan province, eligibility number: SCXK 2020-030.

CTMB drug substance was prepared as in example 1.

TK-X07 was prepared according to the method of example 2.

The edaravone right borneol injection concentrated solution was purchased from the pharmaceutical industry of Miao medicine Co., ltd (specification: 10mg:2mL; lot number: 1811283).

1.2 mechanical impact method for establishing TBI rat model

Preoperative rats were fasted for 12h, rats were anesthetized with 4% isoflurane induction and anesthesia was maintained with 2% isoflurane. Fixing in supine position, and maintaining animal body temperature at about 37deg.C. The prone position fixes the rat on the stereotactic apparatus, cuts off the hair on the top of the head, and disinfects conventionally. A circular bone window with the diameter of 5.0mm is formed at the position 2.0mm beside the right side of the midline and 1.5mm from the front of the herringbone suture by cutting the center of the cranium top by about 1.0 cm. Under the guidance of a stereotactic instrument, a 40g weight is lowered from a position with the height of 15cm to form a free falling body to fall, and the weight is hit on a firing pin with the diameter of 3nm on a brain bone window (the length of the firing pin exposed out is about 3-4 nm), so that the rat cranium can smash a round hole with the diameter of about 3 mm. After beating, removing the firing pin at the wound after 1min, and stopping bleeding and suturing the wound. After the operation, the rats are placed in corresponding cages to control the temperature (24+/-0.5 ℃), and after the anesthesia is awake, the rats are normally bred.

1.3TBI rat model enrollment criteria

After the rats are anesthetized and awake 2 hours after the operation, the model rats are scored by referring to the mNSS nerve function score, and the rats with the score of 8-13 minutes are selected for the next test. The mNSS neurological scoring criteria are shown in Table 1.

TABLE 1mNSS neurological function score

1.4 Experimental grouping

And judging whether the molding is successful or not by using a mNSS score 1h after mechanical impact, and taking the rat with successful molding for the next test.

Rats successfully molded were randomly divided into 6 groups of 12 rats each, each:

sham surgery group (P group): administering an equivalent volume of physiological saline to the TK-X07 high dose group;

model group (T group): administering a blank emulsion of equal volume to the high dose group of emulsion injection;

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

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

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

positive control group (Y group): administering a dose of 2mg/kg of edaravone right borneol injection concentrated solution group;

1.5 neurological scoring

2 hours after mechanical impact, the above 6 groups are all administrated by intraperitoneal injection, and the administration is carried out 1 time a day. After 7d of administration, the nerve functions of each group of rats are comprehensively evaluated by using mNSS score, the motor, the sensation, the climbing and the limb symmetry of the rats are evaluated, the score ranges from 0 to 18 minutes, the higher the score is, the heavier the nerve function damage is, and the scoring is independently completed by unknowns not participating in modeling and administration. The scoring results are shown in table 2.

Table 2 rat short term neurological deficit score

Note that: in comparison with the sham operation (group P), ^### P<0.001; comparison with model group (T group), P<0.01，**P<0.01，*P<0.05; compared with the positive drug control (group Y), ^& P<0.05。

as can be seen from Table 2, the model group (T group) showed a significant increase in mNSS score of 7d after TBI as compared to the sham operation group (P group) ^### P<0.001 Indicating that the TBI rat model has obvious neurological deficit; the CTMB administration (L, M, H group) and the positive drug control group (Y group) with different dosages can obviously reduce mNSS score and improve the neurological deficit caused by TBI, wherein the L, M, H group administration curative effect is better than the Y group, and the M group drug effect is obviously better than the Y group [ ] ^& P<0.05)。

Experimental example 2

Neuroprotection of CTMB on TBI rat model

2.1 evaluation of Secondary epileptic incidence

The experimental materials and the molding mode are the same as those of the effect example 1, and the grouping conditions are as follows:

sham surgery group (P group): administering an equivalent volume of physiological saline to the CTMB group;

model group (T group): administering a blank emulsion of equal volume to CTMB group;

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

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

positive control group (Y group): the edaravone right camphene injection concentrated solution with the dosage of 2mg/kg is given.

2h after TBI molding, intraperitoneal injection administration was performed according to each group of administration schemes, and the secondary epileptic occurrence rate in rats in 48h was observed, and the results are shown in Table 3.

TABLE 3 incidence of acute phase epilepsy in TBI rats

As can be seen from Table 3, the epileptic incidence in the T group of rats was 41.6% in 48h, while the epileptic incidence in both L, M, H and Y group of rats was reduced, indicating that CTMB was able to reduce the secondary epileptic incidence in the TBI rats in 48 h.

2.2 evaluation of survival

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

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

2h after TBI molding, intraperitoneal injection administration was performed according to each group administration schedule, and administration was continued for 10 days, once daily, and the survival rate of rats was observed and recorded for 10 days, and the results are shown in Table 4.

TABLE 4 Long term survival of TBI rats

As can be seen from Table 3, the survival rate in group T10 d was only 60%, while groups L, M, H and Y increased the survival rate in TBI rat 10 d.

2.3 assessment of New Environment adaptability and emotional anxiety

Open field experiments can be used to assess emotional anxiety in a TBI rat model. After the end of the life cycle observation record, the open field experiments were performed on days 10 and 20 after TBI molding, and the dosing group was selected as the medium dose group (group M) for the experiments. The open field is a square area, and the length and the width are 100cm. Rats were released in the indicated areas following the open field experimental guidelines (Smith DF. Biogenic amines and the effect of short term lithium administration on open field activity in rates. Psychopharmacology 1975;41 (3): 295-300.Doi:10.1007/BF00428940.PMID: 125430.). The movement track, movement distance, movement time and other data of the rat in different areas are recorded by a computer tracking system (Noldus Ethovision, tacoma, WA, USA).

As shown in fig. 1, the movement distance of the T group is obviously reduced compared with that of the P group, and the movement distance of the M group and the Y group is obviously higher than that of the T group; the time of movement of each group in the central region is shown in fig. 1 (right), the number of shuttling times and the mobility of the T group in the central region are significantly reduced compared with those of the P group, and the number of shuttling times and the mobility of the M group in the central region are significantly higher than those of the T group. In conclusion, CTMB administration significantly improved the ability of TBI rats to adapt to the new environment and alleviated emotional anxiety.

(note: compared to sham surgery (group P), ^# P<0.01; comparison with model group (T group), P<0.01，**P<0.01，*P<0.05)。

Experimental example 3

Research on action mechanism of CTMB for treating traumatic craniocerebral injury

Experimental materials:

evan blue (C11891158, shanghai Michelin Biochemical technology);

formamide (20190716, tianjin Bodi chemical);

IL-1 detection kit (E-EL-H0149 c, available from WU.S. Yi Rui Te Biotech Co., ltd.);

IL-6 detection kit (E-EL-H2518 c, available from Wohan Iretto Biotech Co., ltd.);

TNF-alpha assay kit (9680019151122, eboltak biotechnology);

GABA detection kit (ZD 0342B47674, WUM Iretto Biotech Co., ltd.);

GLT-1 detection kit (Apr 2023, limited Biotechnology of Rui Xin, quanzhou).

3.1 blood brain barrier permeability assay

The experimental groupings and dosing regimens were as follows:

medium dose group (M group): TK-X07 was administered at a dose of 30 mg/kg.

2h after TBI molding, the rats of each group are injected and administrated intraperitoneally, and after 24h, 4% Evan's blue solution (2.5 mL/kg) is injected into the tail vein of the right side of the rats, and after 1h, the rats are deeply anesthetized, 100mL of physiological saline is infused through the heart, the head is rapidly broken, the brain is taken, and the rats are immediately divided into a left hemisphere, a right hemisphere, a cerebellum and brainstem. After weighing brain tissue wet weight, soaking in 10 times of pure formamide, incubating at 60 ℃ for 48h, centrifuging at 25 ℃ for 30min at 10000rpm/min, absorbing supernatant, detecting Evan blue dye at 622nm by ultraviolet spectrophotometry, drawing a standard curve for quantification, and finally displaying the result as the Evan blue content (mug/g) per gram of brain tissue. The results are shown in fig. 2 (n=6), where the bleeding side hemispheres of the brains of the model (T) had increased blood brain barrier permeability 24h post-operatively compared to the sham (P) and the dose in TK-X07 (M) significantly reduced evans blue exudation and improved blood brain barrier integrity.

3.2IL-1, IL-6 and TNF-alpha assays

Experimental groupings and dosing regimens were as described under 3.1. And (3) performing intraperitoneal administration on different groups of rats 2h after TBI molding, performing deep anesthesia on the rats for 24h after administration, taking out brains after head breakage, taking out about 60-120 mg of cerebral cortex on blood side, adding 10 times of ice-cooled centrifugal buffer solution for biochemical detection, homogenizing for 10min in ice bath, centrifuging at 14000rpm/min at 4 ℃, and taking supernatant to detect the contents of inflammatory factors IL-1, IL-6 and TNF-alpha according to the instructions of the IL-1, IL-6 and TNF-alpha content detection kit. As shown in fig. 3 (n=5 to 8), compared with the P group, the content of IL-1, IL-6 and TNF- α in the bleeding side hemispheres of the T group rats was significantly increased 24h after the operation, while the M group significantly reduced the content of pro-inflammatory cytokines TNF- α, IL-1 and IL-6 in the cerebral cortex tissue of the TBI rat, which was beneficial for alleviating neuroinflammation and improving the motor function of the rats.

3.3GLT-1 and GABA content determination

Experimental groupings and dosing regimens were as described under 3.1. After each group was continuously administered for 7d, the rats were deeply anesthetized, the brains were broken, about 60-120 mg of the hemispheric cortex of the blood side brain was removed, 10 volumes of ice-cold centrifugation buffer for biochemical detection was added, homogenized for 10min under ice bath, centrifuged at 4℃14000rpm/min for 30min, and the supernatants were taken to detect GLT-1 and GABA content, respectively, according to the GLT-1 and GABA content detection kit instructions. Glutamate transporter 1 (GLT-1) is an important glutamate transporter in the brain and can transport extracellular glutamate into the cell; gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter, and a steep increase in extracellular glutamate concentration in pathological conditions leads to neuronal hyperexcitatory toxicity. GLT-1, GABA levels and ratios of the two, as measured in different dosing groups, were used to assess excitatory neurotoxic effects resulting from TBI damage. As shown in fig. 4 (n=6), compared with group P, the bleeding side hemispheres of rats in group T had significantly increased GLT-1 content and GLT-1/GABA ratio at 7 days post-surgery, whereas administration of group M reduced the GLT-1 content, increased GABA content in the brains of TBI rats, significantly reduced the GLT-1/GABA ratio, restored the balance of Excitatory Amino Acids (EAA) and Inhibitory Amino Acids (IAA) in the brains, reduced the excitatory neurotoxicity caused by excess glutamate, and further improved motor function in rats.

Experimental example 4

Protection of mechanical scratch damaged neurons by CTMB

4.1 experimental materials:

HT22 cell lines were purchased from the marsupenario life technologies Co., ltd;

CCK8 (C12029690, sigma-Aldrich, usa);

DMEM high sugar medium (AG 29301810, hyclone, usa);

fetal bovine serum (20010401, gibco U.S.A.)

PBS powder (WK 173618-1, beijing Zhongshan mountain bridge Biotechnology Co., ltd.);

DMSO (20201220, beijing Soy Bao technology Co., ltd.).

4.2 Experimental procedure

Preparation of complete medium: DMEM high sugar medium, fetal bovine serum and penicillin-streptomycin solution (diabody) were mixed at 90:9:1, and preserving in a refrigerator at 4 ℃.

Preparation of serum-free Medium: DMEM high sugar medium and penicillin-streptomycin solution (diabody) were mixed at 99:1, and preserving in a refrigerator at 4 ℃.

With complete mediumHT22 cells were cultured at 1X 10 when they were in logarithmic growth phase ⁴ Each 100. Mu.L/well was inoculated into a 96-well plate, and the edge wells were filled with sterile PBS, 37℃and 5% CO ₂ Culturing for 24h until the cells are completely adhered; the supernatant fluid is discarded and the liquid is discharged,

drug administration group: 100. Mu.L of CTMB diluted in serum-free medium at various concentrations (to give final concentrations of 0.3. Mu.M, 0.625. Mu.M, 1.25. Mu.M, 2.5. Mu.M, respectively) was added. Culturing for 2 hr, and dividing into three groups, and establishing mechanical scratch damage model at 37deg.C and 5% CO ₂ Culturing was continued for 24 hours.

Control group: the same volume of serum-free medium was added for culture without scratch damage, and the rest of the procedure was the same as for the dosing group.

Model group: the same volume of serum-free medium is sequentially added to perform culture after mechanical scratch damage, and the rest operation is the same as that of the administration group.

Subsequently, 810. Mu.L of CCK was added to each well at 37℃with 5% CO ₂ Culturing for 40min, and detecting OD value at 570nm by enzyme labeling instrument, and the result is shown in FIG. 5.

As shown in fig. 5, the model group caused apoptosis or necrosis of HT22 cells due to mechanical scratch damage, and the proliferation activity was significantly reduced, compared to the control group (cell proliferation activity was 1.0); on the other hand, CTMB concentrations of 0.3 μm to 2.5 μm significantly improved the decrease in cell proliferation activity due to mechanical scratch damage, suggesting that it can reduce apoptosis and necrosis due to mechanical damage.

From the above examples, the present invention provides the use of CTMB in the preparation of a medicament for treating or preventing traumatic brain injury diseases.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

The application of CTMB in preparing medicines for treating and/or preventing traumatic craniocerebral injury diseases is provided, wherein the structural formula of the CTMB is shown as formula I:
2. the use of CTMB according to claim 1 for the manufacture of a medicament for the treatment and/or prevention of traumatic brain injury diseases, including secondary epilepsy, neurological or motor dysfunction due to traumatic brain injury, the route of administration of the medicament comprising injection, oral administration, transdermal, inhalation, mucosal administration or subcutaneous implantation.
3. The use of CTMB according to claim 1 for the manufacture of a medicament for the treatment and/or prevention of traumatic brain injury disease, wherein the medicament is an injection.
4. The use of CTMB according to claim 1 for the manufacture of a medicament for the treatment and/or prevention of traumatic brain injury diseases, wherein the injection is a emulsion injection.
5. The use according to claim 1, wherein,

the ratio of the effective dosage of the effective component CTMB in the emulsion injection to the unit mass of human body is 0.2 mg-4.0 mg/kg;

the ratio of the effective dosage of the effective component CTMB in the emulsion injection to the unit mass of the rat is 20 mg-40 mg/kg.
6. A CTMB emulsion injection for treating and/or preventing traumatic brain injury diseases, characterized by comprising the following components in percentage by weight: 0.5 to 5 percent of CTMB, 5 to 30 percent of oil phase, 0.6 to 1.8 percent of emulsifier, 0.001 to 0.01 percent of pH regulator and the balance of water.
7. The emulsion injection according to claim 4, further comprising 0% to 2.5% glycerol.
8. The emulsion injection according to claim 4, wherein the oil phase is selected from one or more of soybean oil, medium chain triglycerides, fish oil, olive oil, structural triglycerides;

the emulsifier is one or more selected from egg yolk lecithin, soybean lecithin, pluronic F68 and polyethylene glycol stearic acid-15 (Solutol HS 15).
9. A method of preparing an emulsion according to any one of claims 5 to 8, comprising the steps of:

(1) Under the protection of nitrogen or inert gas, dissolving CTMB in an oil phase preheated to 70-80 ℃, and then dissolving an emulsifier in the oil phase dissolving CTMB or in a water phase at 70-80 ℃;

(2) Mixing the oil phase and the water phase by high-speed shearing to prepare colostrum, and regulating the pH value;

(3) Homogenizing the colostrum under high pressure for 1-3 times until the average particle diameter of emulsion drops is less than or equal to 0.4 mu m, filtering, and performing rotary hot-press sterilization to obtain the emulsion injection containing CTMB.