CN117695261A - Application of CTMB in preparation of medicines for treating and/or preventing ischemic cerebral apoplexy - Google Patents

Application of CTMB in preparation of medicines for treating and/or preventing ischemic cerebral apoplexy Download PDF

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CN117695261A
CN117695261A CN202311776583.2A CN202311776583A CN117695261A CN 117695261 A CN117695261 A CN 117695261A CN 202311776583 A CN202311776583 A CN 202311776583A CN 117695261 A CN117695261 A CN 117695261A
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ctmb
injection
emulsion injection
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emulsion
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毛声俊
廖灿
严睿洁
李蕊
沈千万
张检
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Chengdu Xinrui Taikang Technology Co ltd
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Chengdu Xinrui Taikang Technology Co ltd
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Abstract

The invention provides an application of CTMB in preparing a medicament for treating and/or preventing ischemic cerebral apoplexy, an emulsion injection for treating the ischemic cerebral apoplexy and a preparation method thereof, and provides a medicament for remarkably improving the nerve behavior, the sense and the motor function of a rat in the ischemic cerebral apoplexy for treating or preventing the ischemic cerebral apoplexy, which is effective, safe and has no obvious toxic or side effect.

Description

Application of CTMB in preparation of medicines for treating and/or preventing ischemic cerebral apoplexy
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an application of CTMB in preparing medicines for treating and/or preventing ischemic cerebral apoplexy.
Background
Stroke has become the second leading cause of death worldwide and the third leading cause of disability, with over 80% of patients being ischemic stroke. Ischemic stroke is also called cerebral infarction, is brain injury caused by partial or complete brain tissue blood flow limitation, and has the characteristics of high morbidity, high mortality, high disability rate and high recurrence rate. Ischemic cerebral apoplexy can cause serious clinical symptoms such as hemiplegia, strabismus, aphasia, dysesthesia, consciousness reduction and the like, seriously influence the quality of life and cause great burden to families and society.
The occurrence and development of ischemic stroke involves multiple complex pathological mechanisms such as oxidative stress injury, mitochondrial dysfunction, apoptosis, inflammatory response, and blood brain barrier injury. When ischemic stroke occurs, interruption of cerebral blood supply causes cerebral hypoglycemia and hypoxia, intracellular ATP energy exhaustion is induced, energy-dependent ion transport dysfunction, depolarization of neurons and glial cells, calcium ion influx causes release of excitatory amino acids such as glutamic acid, and excitatory toxicity effect is generated. A large amount of calcium ions in the cells activate metabolism-related enzymes to generate a large amount of nitric oxide, arachidonic acid metabolites, oxygen free radicals and the like, induce oxidative stress injury in the brain, and further trigger apoptosis and necrosis of nerve cells. Restoring cerebral blood flow in a short period of time can alleviate cerebral ischemic injury, but reperfusion time exceeds a certain time limit, possibly aggravating brain injury. This is because free radical generation is further increased during reperfusion, exacerbating neuronal death. Necrotic cells release damage related model molecules (DAMP) to promote chemotaxis of inflammatory cells, increase the generation of stimulation signals such as inflammatory factors, chemokines and ROS, and promote immune cell infiltration, thereby exacerbating cerebral ischemia damage and blood brain barrier destruction. On the other hand, inflammatory factors activate matrix metalloproteinases in the brain, which results in increased permeability of the blood brain barrier, aggravates the condition of the cerebral apoplexy patient, and seriously affects the prognosis of the patient.
The current common medicines for clinically treating ischemic cerebral apoplexy mainly comprise recombinant tissue plasminogen activator (rt-PA), neuroprotection agent and the like, and the rt-PA is the most common medicine for treating ischemic cerebral apoplexy. However, rt-PA has a narrow clinical window of administration and may increase the risk of cerebral hemorrhage, inducing adverse reactions. The neuroprotectant can reduce neuronal death after cerebral ischemia, improve the tolerance of nerve cells to ischemia and hypoxia, promote the recovery of the nerve function of a patient, and improve the prognosis of the patient, and the neuroprotectant clinically used at present mainly comprises free radical scavengers edaravone, GABA receptor agonists piracetam and the like. However, most of the marketed neuroprotective agents are single-target drugs, and often act on the downstream and middle links of secondary nerve injury caused by stroke, and clinical efficacy is limited. Therefore, the development of a new medicine capable of effectively treating ischemic cerebral apoplexy has important clinical value and social significance.
Disclosure of Invention
Therefore, the invention aims to avoid the defects of the prior art and provide the application of CTMB in preparing the medicines for treating and/or preventing ischemic cerebral apoplexy, and the medicines for remarkably improving the nerve behaviors and the sensorimotor functions of ischemic cerebral apoplexy rats are effective, safe and have no obvious toxic and side effects.
The above object of the present invention is achieved by the following technical measures:
the invention provides an application of CTMB in preparing medicines for treating and/or preventing ischemic cerebral apoplexy, wherein the structural formula of the CTMB is shown as formula 1
Preferably, the route of administration of the drug includes injection, oral, transdermal, inhalation, mucosal administration or subcutaneous implantation.
Preferably, the medicament is an injection.
Preferably, the injection is a emulsion injection.
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).
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 10 mg-20 mg/kg.
The invention also provides a preparation method of the emulsion injection, which is characterized by comprising 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 nerve behavior function of rats with ischemic cerebral apoplexy and recover the sensory and motor functions of the rats;
(2) Effective and safe, and has no obvious toxic and side effects.
Detailed Description
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.
The invention provides an application of CTMB in preparing a medicament for treating and/or preventing ischemic cerebral apoplexy, wherein the structural formula of the CTMB is shown as formula 1
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 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.
In the present invention, the emulsion injection comprises CTMB 0.5% to 5%, preferably 0.5% to 2%, more preferably 0.5%;
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);
the emulsion injection in the invention comprises pH regulator 0.001% -0.01%, preferably 0.005; 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 glycerol 0% to 2.5%, preferably 2.0% to 2.5%, further preferably 2.25%, 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 10 mg-20 mg/kg;
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.
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 autoclave sterilization, the temperature is preferably 121 ℃, the time is preferably 12 minutes, and the pressure is preferably 103.4kPa.
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 plant),
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 reagent Co., ltd.),
Anhydrous magnesium sulfate (Q/12 KM3936-2019, tianjin chemical reagent Co., ltd.),
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. Dissolving the above obtained liquid in 100mL anhydrous dichloromethane, adding trichloro at 0deg.CAluminum was formed, warmed to room temperature, stirred for 2.5 hours, then quenched with 300mL of water, extracted with ethyl acetate (3×200 mL), the organic phases were combined, dried over anhydrous magnesium sulfate, 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 5.0g of CTMB and 100.0g of soybean oil for injection, placing 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, regulating the pH to 8.5-11.0 by sodium hydroxide, and adding water to 1000mL to prepare the colostrum. Homogenizing the primary emulsion for 2 times by using a high-pressure homogenizer to ensure that the average particle size of the homogenized emulsion drops is not more than 0.4 mu m, filling the emulsion into a 5mL glass ampoule under the protection of nitrogen filling, and performing hot press sterilization at 121 ℃ for 12min to obtain TK-X07, wherein the concentration of CTMB is 5mg/mL. A blank emulsion injection without CTMB was prepared in the same manner.
Example 3
CTMB is effective in treating acute phase of cerebral ischemic stroke of rats caused by the suppository method.
3.1 experimental materials: SPF SD rats (male, weight 200-230 g, purchased from Sichuan Daos laboratory animal Co., ltd., qualification number: SCXK 2020-0030),
MCAO bolt line (from Beijing West strong tech Co., ltd., model 2432-A5)
CTMB drug substance is prepared in example 1 (lot number: 20221107), and emulsion injection is prepared in example 2 (lot number: 20221114),
Edaravone right camphol injection concentrated solution (purchased from Miao pharmaceutical Co., ltd. (specification: edaravone 10mg/5mL, right camphol 2.5mg/mL; lot number: 180-220522)).
3.2 experimental grouping: and (5) judging whether the modeling is successful or not by using a Zea Longa score 2 hours after MCAO (MCAO) operation, and taking rats with successful modeling for random grouping and administration.
Rats were randomly divided into Sham-operated group (Sham group, administration of a blank emulsion of the same volume as TK-X07 high dose group), model group (Vehicle group, administration of a blank emulsion of the same volume as TK-X07 high dose group), TK-X07 low dose group (CTMB-L group, 10 mg/kg), TK-X07 high dose group (CTMB-H group, 20 mg/kg), and edaravone right camphol concentrated solution for injection (EDB group, commercially available, 3 mg/kg), 10 groups each were administered by intraperitoneal injection. ( It should be noted that the administration mode of rats in the experiment is intraperitoneal injection, the ratio of the effective amount of CTMB in the emulsion injection to the unit mass of human body is 0.2 mg-4.0 mg/kg, and the ratio of the effective amount of CTMB in the emulsion injection to the unit mass of human body during intravenous drip is calculated from the effective amount of rats. Considering the bioavailability of the medicine, the peak concentration and the peak time of the medicine, which are different from each other due to different species and different administration modes, the proportion of the effective component CTMB in the emulsion injection to the unit mass of human body is finally determined to be 0.2 mg-4.0 mg/kg through conversion )
3.3 line plug method for establishing ischemia reperfusion model
Preoperative rats were fasted for 12 hours and the rats were anesthetized with 10% chloral hydrate (350 mg/kg, i.p.). Fixing in supine position, and maintaining animal body temperature at about 37deg.C. The neck is prepared, a median incision of the neck is taken, muscles and fascia are separated along the inner margin of the sternocleidomastoid muscle, the right side is exposed, the Common Carotid Artery (CCA), the External Carotid Artery (ECA) and the Internal Carotid Artery (ICA) are passively separated, and the treatment lines are reserved at the proximal end of the CCA, the ICA and the ECA. Ligating the proximal end of the CCA, ECA, temporarily clamping ICA with an arterial clip, then poking a small hole with a needle at the position about 4mm from the bifurcation part of the CCA, inserting a bolt line into the ICA through the CCA, loosening the arterial clip on the ICA, slowly pushing the bolt line, tightly tying a thin line at the ICA when the mark on the bolt line reaches the bifurcation part and slightly feels resistance, cleaning the wound surface with normal saline, and suturing. The procedure was the same as in the experimental group except that no plug was inserted in the sham operation. After anesthesia and wakening, normal feeding is performed.
3.4 cerebral ischemia model selection criteria
Referring to the Zea Longa neurological function score (Longa EZ, weinstein PR, carlson S, cummins R.reverse middle cerebral artery occlusion without craniectomy in rates. Stroke.1989Jan;20 (1): 84-91.Doi:10.1161/01. Str.20.1.84.), rats were scored 2 hours after anesthesia and wakefulness, scoring 1-2 points into groups:
0 point: no symptom of nerve function deficiency and normal activity;
1, the method comprises the following steps: the contralateral forepaw cannot be fully extended;
2, the method comprises the following steps: the animals turn round when crawling;
3, the method comprises the following steps: the body is inclined to the hemiplegia side;
4, the following steps: and the patient can not walk spontaneously and the consciousness is lost.
3.5 short term neurological deficit scoring
After 24 hours of modeling, the nerve function of the rat is comprehensively evaluated by adopting improved mNSS (common-node-surface-less-system) scores, wherein the mNSS scores are shown in table 1, the Garcia scores are shown in table 2, and the motor, the sensation, the climbing and the limb symmetry of the rat are evaluated, wherein the mNSS scores range from 0 to 18 minutes, and the higher the score is, the heavier the nerve function damage is; the Garcia score ranges from 3 to 18 points, the lower the score, the more the neurological impairment. Scoring was done independently by unknowns not involved in modeling and administration.
TABLE 1mNSS neurological function score
TABLE 2Garcia neurological score
As can be seen from table 3, the model group (Vehicle group) showed a significant increase in mNSS score (P < 0.01) 24 hours after surgery and a significant decrease in Garcia score (P < 0.01) compared to the Sham group (Sham group), and it was seen that the model group rats developed a significant neurological deficit 24 hours after MCAO surgery; different doses of CTMB administration (CTMB-L, CTMB-H group) and edaravone right camphene injection concentrated solution administration (EDB group) can reduce mNSS score and increase Garcia score to different degrees, and improve neurological function defect caused by MCAO, wherein the effect of the CTMB-H group administration is most remarkable (P < 0.01), and the effect of the CTMB-H group administration is superior to that of the edaravone right camphene which is a medicament clinically used for improving ischemic cerebral apoplexy. During the course of the experiment, no toxic side effects associated with CTMB administration were observed.
Table 3 rat short term neurological deficit score
Note that: compared to Sham surgery (Sham group), ## P<0.01; compared with the model group (Vehicle group), P<0.01，*P<0.05。
Example 4
CTMB has long-term therapeutic effect on rat ischemic stroke caused by rat wire embolism.
4.1 neurological deficit scoring
Experimental materials, groups (n=10 to 15), molding modes and scoring criteria were treated in the same short term, and 2 hours after molding by the wire suppository method, administration was immediately according to the group administration regimen, and thereafter continued for 14 days, once daily, and modified mNSS scores were performed on days 1, 4, 7, and 14.
As can be seen from table 4, the model group (Vehicle group) showed a significant increase in mNSS score (P < 0.01) at 14 days after surgery compared to the Sham operation group (Sham group), and it was seen that rats in the model group developed a significant neurological deficit at 14 days after MCAO operation; the mNSS score can be reduced to different degrees in the CTMB administration (CTMB-H group) and the edaravone right camphene injection concentrated solution group (EDB group) within 1-7 days, and the neurological defect caused by MCAO is improved, wherein the effect of the CTMB-H group administration is most remarkable, and the curative effect is slightly better than that of the edaravone right camphene injection concentrated solution for clinical medicines for improving ischemic cerebral apoplexy. And on day 14, CTMB-H group still significantly reduced mNSS scores. During the course of the experiment, no toxic side effects associated with CTMB administration were observed.
Table 4 long term neurological deficit score in rats
Note that: compared to Sham surgery (Sham group), # P <0.01; compared to the model set (Vehicle set),
**P<0.01，*P<0.05。
4.2 adhesion experiments
Before molding, the rats are subjected to one week of adhesion experiment training, and the rats with adhesive tapes which can be successfully torn off within 10 seconds are screened out for molding. Immediately following the wire-plug molding, dosing was performed according to a group dosing regimen, followed by a further 14 days of dosing, once daily, and adhesion experiments were performed on days 1, 4, 7, 10 and 14 to evaluate sensory and motor nerve function in rats. The specific operation is as follows: circular tape of approximately 12mm diameter was adhered to the left forelimb sole of the rat and the time to feel and remove the tape was recorded. If the mice were unable to perceive and/or remove the tape within 60 seconds, it was recorded as 60 seconds.
As can be seen from table 5, the model group (Vehicle group) showed a significant increase in time to the perceived tape (P < 0.05) at 14 days post-surgery compared to the Sham group (Sham group), and the model group rats had a reduced left forelimb perception at 14 days post-MCAO; different doses of CTMB and EDB can promote the restoration of the cognitive ability of rats.
TABLE 5 time for rat to perceive tape
Note that: compared to Sham surgery (Sham group), ## P<0.01， # P<0.05; compared to the model set (Vehicle set),
**P<0.01，*P<0.05。
as can be seen from table 6, the model group (Vehicle group) significantly increased the tape drop time (P < 0.01) 14 days after surgery compared to the Sham operation group (Sham group), and the model group rats had a reduced left forelimb locomotion ability 14 days after MCAO operation; the time (P < 0.05) for the rats to drop the adhesive tape can be obviously reduced from the 4 th day of the CTMB-L group, the time (P < 0.05) for the rats to drop the adhesive tape can be obviously reduced from the 10 th day of the CTMB-H group, and the efficacy of the medicine is superior to that of edaravone right camphene which is a medicine clinically used for improving ischemic cerebral apoplexy. During the course of the experiment, no toxic side effects associated with CTMB administration were observed.
TABLE 6 time for the rats to drop the tape
Note that: compared to Sham surgery (Sham group), ## P<0.01， # P<0.05; compared with the model group (Vehicle group), P<0.01，*P<0.05。
In conclusion, the long-term administration of CTMB can obviously promote the recovery of the motor function of rats and can recover the perception capability of rats to a certain extent.
Example 5
Therapeutic effects of CTMB on rat models of ischemic stroke due to photochemical embolism by long-term administration.
5.1 experimental materials: SPF-grade SD rats. Male, weight 200-230 g (from Sichuan Dashuo laboratory animal Co., ltd., qualification number: SCXK 2020-0030)
Rose red (purchased from sigma company in the united states)
CTMB drug substance is self-made in example 1 (lot number: 20221107), and emulsion injection is self-made in example 2 (lot number: 20221114)
5.2 experimental grouping: after the rats were anesthetized and awake, a preliminary evaluation was performed. The molding success criteria are as follows: (1) the anterior limb or the posterior limb buckling (2) after moulding cannot normally run straight (3) the head deviates from the vertical axis by more than 10 degrees within 30s (4) and falls to the paraplegia side. Any one of the 4 items is successful in molding. Rats successfully modeled were randomly dosed.
Rats were randomly divided into Sham-operated group (Sham group, given the same volume of blank emulsion as TK-X07 high-dose group), model group (Vehicle group, given the same volume of blank emulsion as TK-X07 high-dose group), TK-X07 low-dose group (CTMB-L group, 10 mg/kg), TK-X07 high-dose group (CTMB-H group, 20 mg/kg), each of 12 groups were administered by intraperitoneal injection.
5.3 photochemical embolism method for constructing ischemic cerebral apoplexy model
Preoperative rats were fasted for 12 hours, induced for anesthesia with 4% isoflurane, and prone position was fixed in brain stereotactic apparatus with 2% isoflurane maintained for anesthesia. After the skin of the head and the head is disinfected by iodophor, the skin is longitudinally cut, then the skull is exposed, the connective tissue membrane on the surface of the skull is peeled off, the bregma point (0, 0) is taken as the basic origin, 3.5mm is arranged on the right side of bregma, 0.5mm (AP: 0.5mm, ML:3.5 mm) is arranged under the front halogen as the center, a bone window with the diameter of 6mm is manufactured by a dental drill, the dura mater is not damaged, and physiological saline is dripped until the blood vessels on the surface of the brain are clear. Then, the film was irradiated with a yellow-green laser beam having a diameter of 8mm for 20 minutes. The sham operation is carried out except that the rats are not irradiated by a laser at fixed points and are not injected with rose bengal, and the rest operation steps are the same as those of the experimental group. After anesthesia and wakening, normal feeding is performed.
5.4 neurological deficit scoring
The scoring criteria were identical to mNSS scoring criteria under 1.3. The administration was immediately followed by a divided dosing regimen 2 hours after photochemical embolism modeling, and continued for 10 days thereafter, once daily, with modified mNSS scores on days 1, 3, 5, 7 and 10.
As can be seen from table 7, the model group (Vehicle group) showed a significant increase in mNSS score (P < 0.01) at 10 days after surgery compared to the Sham operation group (Sham group), and the model group rats developed a significant neurological deficit at 10 days after surgery; the administration of CTMB (CTMB-L, CTMB-H group) with different doses for 1-10 days can reduce mNSS score to different degrees, and improve neurological deficit caused by photochemical embolism, wherein the administration effect of the CTMB-L group is most remarkable. During the course of the experiment, no toxic side effects associated with CTMB administration were observed.
TABLE 7 rat mNSS neurological function score
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Note that: prosthetic operation(Sham group) comparison, ## P<0.01， # P<0.05; compared with the model group (Vehicle group), P<0.01，*P<0.05。
5.5 adhesion test
Before molding, the rats are subjected to one week of adhesion experiment training, and the rats with adhesive tapes which can be successfully torn off within 10 seconds are screened out for molding. Immediately after 2 hours after molding, administration was performed according to a group administration regimen, and administration was continued for 5 days thereafter, once daily, and adhesion experiments were performed on days 1, 3, and 5 to evaluate the sensory and motor functions of rats. The specific operation is as follows: circular tape of approximately 12mm diameter was adhered to the left forelimb sole of the rat and the time to feel and remove the tape was recorded. If the mice were unable to perceive and/or remove the tape within 60 seconds, it was recorded as 60 seconds.
As can be seen from table 8, the model group (Vehicle group) showed a significant increase in the time to perceive the adhesive tape 5 days after surgery (P < 0.01) compared to the Sham group (Sham group), and the model group rats left forelimb perception was seen to be diminished 5 days after MCAO surgery; low doses of CTMB significantly promote recovery of the cognitive ability of rats from day 3.
TABLE 8 time for rat to perceive tape
Note that: compared to Sham surgery (Sham group), ## P<0.01， # P<0.05; compared to the model set (Vehicle set),
**P<0.01，*P<0.05。
as can be seen from table 9, the model group (Vehicle group) significantly increased the time to tape drop (P < 0.01) 5 days after surgery compared to the Sham operation group (Sham group), and the model group rats left forelimb movement ability was seen to be reduced 5 days after MCAO operation; low doses of CTMB significantly promote recovery of motor ability in rats from day 3.
TABLE 9 time for the rats to drop the tape
Note that: compared to Sham surgery (Sham group), ## P<0.01， # P<0.05; compared to the model set (Vehicle set),
**P<0.01，*P<0.05。
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 (9)

  1. The application of CTMB in preparing the medicines for treating and/or preventing ischemic cerebral apoplexy is characterized in that the structural formula of CTMB is shown as formula 1
  2. 2. The use of CTMB according to claim 1 for the manufacture of a medicament for the treatment and/or prevention of ischemic stroke, wherein the route of administration of the medicament comprises injection, oral administration, transdermal, inhalation, mucosal administration or subcutaneous implantation.
  3. 3. Use of CTMB according to claim 1 or 2 for the preparation of a medicament for the treatment and/or prevention of ischemic stroke, wherein the medicament is an injection.
  4. 4. The use of CTMB according to claim 3 for the preparation of a medicament for the treatment and/or prevention of ischemic stroke, wherein the injection is a emulsion injection.
  5. 5. An emulsion injection for treating ischemic cerebral apoplexy is 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.
  6. 6. The emulsion injection for treating ischemic stroke according to claim 5, wherein the emulsion injection further comprises 0% -2.5% glycerol.
  7. 7. The emulsion injection for treating ischemic stroke as claimed in claim 5, wherein said oil phase is 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).
  8. 8. An emulsion injection for treating ischemic stroke according to any one of claims 5-7, 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 10 mg-20 mg/kg.
  9. 9. A method of preparing an emulsion injection as claimed in 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.
CN202311776583.2A 2023-12-21 2023-12-21 Application of CTMB in preparation of medicines for treating and/or preventing ischemic cerebral apoplexy Pending CN117695261A (en)

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