CN110801451A - Medicine for treating amyotrophic lateral sclerosis and method for improving blood brain barrier opening effect - Google Patents
Medicine for treating amyotrophic lateral sclerosis and method for improving blood brain barrier opening effect Download PDFInfo
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Abstract
The invention relates to the technical field of ultrasound-combined microvesicles, in particular to a medicine for treating amyotrophic lateral sclerosis by applying ultrasound-combined microvesicle technology and a method for improving the blood brain barrier opening effect. The medicine and the method can improve the brain medicine/blood medicine ratio percentage of edaravone entering the brain. In a mouse experiment, the medicine is combined with the ultrasound to open the blood brain barrier of a mouse and deliver edaravone to the brain, after 4 weeks of treatment, the mouse of the ultrasound combined microbubble administration group is 10.57 percent higher than the claw holding force of a model group (p is less than 0.05), the retention time on a rotating rod is 79.8 percent longer than that of the mouse of the model group (p is less than 0.01), and the retention time is 57.3 percent longer than that of the Eda group (p is less than 0.05). The results show that the ultrasound combined with the microvesicles delivers the edaravone into the brain, compared with the traditional administration mode, the brain bioavailability can be improved, and the disease progression of the SOD1 mouse can be more effectively delayed.
Description
Technical Field
The invention relates to the technical field of ultrasound-combined microvesicles, in particular to a medicine for treating amyotrophic lateral sclerosis by applying ultrasound-combined microvesicle technology and a method for improving blood brain barrier opening effect.
Background
Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig's disease, progressive freezing human disease, motor neuron disease, is a progressive and fatal neurodegenerative disease. Amyotrophic lateral sclerosis results from degeneration of motor neurons that control skeletal muscles within the central nervous system. As the upper and lower motor neurons degenerate and die, the patient's muscles gradually weaken and atrophy; finally, the brain loses its ability to control voluntary movements completely; eventually causing speech, swallowing, and respiratory disturbances. ALS usually progresses from morbidity to respiratory paralysis death in 3-5 years. Amyotrophic lateral sclerosis is a rare disease, which is affected by 1-2 out of every 10 million people every year in most countries, and although the prevalence of ALS is low, the rapid mortality rate of the disease is high. In the united states and the uk, one out of every 500 dead adults is due to ALS disease. Currently, only riluzole (riluzole) and edaravone (edaravone) are drugs approved by the FDA in the united states for the treatment of ALS. Riluzole is the first drug approved by the FDA in the united states for the treatment of ALS and its primary mechanism of action is through various routes to inhibit the toxic damage of glutamate to neurons, but it merely delays the progression of ALS. Over 60 drugs have been studied clinically in the last 20 years, most of which have failed due to their potential therapeutic efficacy in ALS. The treatment of ALS requires effective drugs. Edaravone became the second FDA approved ALS therapeutic agent in 2017 in 5 months at 22 years intervals. Edaravone, chemical name 3-methyl-1-phenyl-2-pyrazoline-5-ketone, is an antioxidant drug developed, produced and sold by Mitsubishi of Japan Honda, and is used for scavenging free radicals to treat cardiovascular and cerebrovascular diseases such as ischemic stroke. Animal experiment results show that in mice administered with edaravone, the over-expressed area of SOD1 is reduced, anterior horn of lumbar vertebra motor neurons are well preserved, and the edaravone has good prevention and treatment effects on ALS. Clinical studies have shown that edaravone can delay up to 33% decrease in physical function in patients compared to placebo, as assessed for efficacy by the ALS patient functional rating scale (ALSFRS-R), a clinical standard widely used to assess the functional status of patients with amyotrophic lateral sclerosis.
However, ALS is fatal and cannot be completely cured, and the search for ALS therapeutic drugs is a research hotspot. One problem faced by the need to use drugs to treat ALS is the presence of the Blood-brain barrier (BBB) within the central nervous system. This barrier is a protective barrier for the brain, which can select substances in the blood to enter the brain, and can prevent most toxic substances from entering the brain to stabilize the internal environment of the brain, but it also prevents most drugs from entering the brain, and thus becomes a barrier to the treatment of central nervous system diseases. Recently, low frequency Focused Ultrasound (FUS) in combination with Microbubbles (MBs) has proven to be an effective method for non-invasive, transient, reversible, and local opening of the blood-brain barrier. Aiming at different diseases, the administration effect of a certain drug is closely related to the conditions of the micro-bubble and the ultrasound, and in order to improve the efficiency of edaravone in the brain and the effect of treating ALS, the combined application condition of edaravone, the micro-bubble and the ultrasound needs to be researched.
Disclosure of Invention
Aiming at the problem that the brain entry efficiency and the ALS treatment effect of the existing edaravone administration mode are required to be further improved, the invention provides a medicine for treating amyotrophic lateral sclerosis by applying ultrasonic combined microbubble technology and a method for improving the blood brain barrier opening effect.
In order to achieve the purpose, the invention adopts the following technical scheme.
In a first aspect of the invention, a medicament for treating amyotrophic lateral sclerosis is provided, which comprises a microbubble composition and edaravone, wherein the microbubble composition comprises microbubble liquid and perfluoropropane, and the microbubble liquid comprises foam and hydration liquid;
the foaming body consists of DSPC and DSPE-PEG2000 according to the mass ratio of (8-10) to 1;
the hydration liquid is formed by mixing phosphate buffer saline solution, glycerol and propylene glycol according to the volume ratio of 8: 0.9-1.1: 1;
the content of the foam in the micro-bubble liquid is (2.5-3.5) mg/mL.
Preferably, the foaming body consists of DSPC and DSPE-PEG2000 according to the mass ratio of 9: 1; the hydration liquid is formed by mixing phosphate buffer salt solution, glycerol and propylene glycol according to the volume ratio of 8: 1; the content of the foam in the microbubble liquid was 3 mg/mL.
Preferably, the microbubble composition is horizontally vibrated to form microbubbles with the particle size of 0.4-12 μm, more than 90% of the microbubbles have the particle size of less than 1.81 μm, and the concentration of the microbubbles is greater than or equal to 5.721 x 109one/mL.
Preferably, the ratio of the volume of the microbubble liquid to the volume of the perfluoropropane is 1: 4.
Preferably, the microbubble composition is contained in a sealed container, and the pressure inside the sealed container is greater than or equal to the pressure outside the sealed container.
Preferably, the ratio of the mass of edaravone to the volume of microbubble composition is (70-80) mg: 1 mL.
More preferably, the mass of edaravone to volume of microbubble composition ratio is 75 mg: 1 mL.
The medicine for treating amyotrophic lateral sclerosis is characterized by being prepared by the following steps:
s1, uniformly mixing phosphate buffer salt solution, glycerol and propylene glycol to obtain a hydration solution;
dissolving DSPC and DSPE-PEG2000 in chloroform to obtain a mixed solution;
s2, evaporating the chloroform in the mixed solution to form a foaming body;
s4, adding a hydration liquid into the foaming body to dissolve the foaming body into the hydration liquid to prepare micro-bubble liquid;
s5, filling the micro-bubble liquid into a sealed container, and then replacing gas above the micro-bubble liquid with perfluoropropane to prepare a micro-bubble composition;
s6, the microbubble composition and the edaravone are combined to form the medicine.
On the other hand, the invention provides a method for improving the blood brain barrier opening effect, after any one of the medicines is injected into the vein, ultrasonic is directionally applied to the cerebral motor cortex on two sides, and the ultrasonic parameters are as follows: the center frequency is 0.970MHz, the sound pressure is 0.63MPa, the repetition frequency is 1Hz, the irradiation duration is 60s, and the duty ratio is 10%.
Compared with the prior art, the invention has the beneficial effects that:
the microbubble composition and the edaravone are combined into the pharmaceutical composition for treating ALS, the pharmaceutical composition is used for opening the blood brain barrier and conveying the edaravone into the brain in combination with ultrasound, and the brain drug/blood drug ratio percentage of the edaravone into the brain can be increased. In a mouse experiment, the medicine is combined with the ultrasound to open the blood brain barrier of a mouse and deliver edaravone to the brain, after 4 weeks of treatment, the mouse of the ultrasound combined microbubble administration group is 10.57 percent higher than the claw holding force of a model group (p is less than 0.05), the retention time on a rotating rod is 79.8 percent longer than that of the mouse of the model group (p is less than 0.01), and the retention time is 57.3 percent longer than that of the Eda group (p is less than 0.05). The results show that the ultrasound combined with the microvesicles delivers the edaravone into the brain, compared with the traditional administration mode, the brain bioavailability can be improved, and the disease progression of the SOD1 mouse can be more effectively delayed.
Drawings
FIG. 1 is a diagram showing the microscopic form (scale: 10 μm) of microbubbles in a microbubble liquid composition prepared according to an example of the present invention;
FIG. 2 is a graph showing the distribution of the sizes of microbubbles in a microbubble liquid composition prepared according to an embodiment of the present invention;
FIG. 3 shows the light mirror and fluorescence imaging of Evans blue into the mouse brain;
FIG. 4 is a statistical graph of the brain drug/blood ratio percentage of edaravone for different modes of administration (n-5, p < 0.05);
figure 5 is a bar graph of paw holding power of WT mice and ALS model SOD1 mice before treatment (×: p < 0.0001);
FIG. 6 is a histogram of paw holding power of groups of mice 4 weeks after treatment (p < 0.05, p < 0.01);
FIG. 7 is a bar graph of data for each group of mice (. p. < 0.05,. p. < 0.01);
FIG. 8 is a graph showing HE staining of lumbar anterior cord motor neurons in each group of mice (scale: 20 μm);
FIG. 9 is a statistical graph of the number of lumbar anterior horn motoneurons in each group of mice (. about.p < 0.01,. about.p < 0.0001);
FIG. 10 shows the microscope and fluorescence imaging of Evans blue into the mouse brain in comparative experiment 1;
fig. 11 shows the light mirror and fluorescence imaging of evans blue into the mouse brain in comparative experiment 2.
Detailed Description
In order to more fully understand the technical contents of the present invention, the technical solutions of the present invention will be further described and illustrated with reference to the following specific embodiments.
The invention aims to open the blood brain barrier by combining low-intensity focused ultrasound and microbubbles, improve the efficiency of delivering edaravone into the brain and the effect of treating ALS, and provide a powerful auxiliary tool for delivering ALS treatment drugs.
Transgenic SOD1 mice are the animal model of ALS that studies the most widespread use of familial ALS. The invention adopts ALS transgenic mice-SOD 1-G93A mice, and the mutant gene is SOD1 (mutant human SOD1 gene driven by endogenous SOD1 promoter, single amino acid with glycine at codon 93 is substituted into alanine). The model is manifested as one or more acroparalysis, a phenotype similar to human amyotrophic lateral sclerosis. Based on the method, ultrasound (0.970MHz, sound pressure of 0.63MPa, repetition frequency of 1Hz, irradiation duration of 60s and duty ratio of 10%) is combined with a microbubble open mouse (13-week-old SOD1 mouse) to deliver edaravone to the brain, the brain-entering content of edaravone is detected, the SOD1-G93A transgenic mouse (SOD1) is subjected to treatment for 6 weeks, ultrasound twice per week is combined with microbubble open blood brain barrier to deliver edaravone to the brain for treatment, grip strength and rotarod experiments are adopted to evaluate the movement capacity of the ALS transgenic mouse, the mouse is subjected to euthanasia treatment after treatment is finished, and the spinal cord and brain of the mouse are subjected to pathological detection to evaluate the treatment effect.
Compared with the simple tail vein injection, the brain drug/blood drug ratio percentage of the ultrasound combined microbubble for opening the blood brain barrier and delivering the edaravone into the brain is improved by 0.43 percent (p is less than 0.05). The detection result shows that compared with wild mice, the SOD1 group mice have a certain decline in the claw holding power and the movement capacity at the initial stage of disease attack (p is less than 0.05); after 4 weeks of treatment, the mice in the group administered with the ultrasound-combined microvesicles were improved by 10.57% (p < 0.05) in the paw grip test compared with the model group, while the mice in the group administered with the ultrasound-combined microvesicles in the tail vein injection test were not statistically different from the model group, and the residence time on the rotor bar was prolonged by 79.8% (p < 0.01) and 57.3% (p < 0.05) in the rotor bar test compared with the Eda group. The results show that the ultrasound combined with the microvesicles delivers the edaravone into the brain, compared with the traditional administration mode, the brain bioavailability can be improved, and the disease progression of the SOD1 mouse can be more effectively delayed.
Specific experiments are shown below.
Experimental animals and groups
Transgenic SOD1 mice (strain: B6.Cg-Tg (SOD 1. G93A)1Gur/J) were purchased from Jackson laboratories, USA, bred by cross breeding with wild type (WT, with the same genetic background as SOD1 mice) female mice (strain: C57BL/6), and positive mice identified by standard PCR techniques.
49 SOD1 female mice, which were 13 weeks old, were randomly divided into five groups, namely, a model group (ALS group, n ═ 9), a simple ultrasound group (FUS group, n ═ 10), an ultrasound microbubble group (FUS/MB group, n ═ 10), a tail vein injection group (Eda group, n ═ 10), and an ultrasound microbubble administration group (FUS/MB + Eda), and 10 WT mice were simultaneously used as negative controls (WT group).
Second, main reagent and instrument
The main reagents and instruments used in the experiment: claw grip tester, rotating rod fatigue tester (huai bei zhenhua bio instruments ltd, china), brain stereotaxic instrument (reward, china), small animal anesthesia apparatus (reward, china), signal generator (AFG3102C, Tektronix, usa), power amplifier (2100L, Electronics & innovations, usa), ultrasonic transducer (sienna boyou ultrasound, china), chromatograph (UltiMate 3000 RS, seimer feier science ltd), mass spectrometer (Q active high resolution mass spectrometer, seimer feier science ltd), evans blue (Regal, china), edala HPLC standards (melphalan, china, > 99%).
Preparation of microbubble composition
The mechanical oscillation method is adopted, and the structure of the microbubble is lipid-encapsulated inert gas perfluoropropane.
The preparation steps of the microvesicle are as follows:
(1) uniformly mixing phosphate buffer salt solution, glycerol and propylene glycol according to the volume ratio of 8: 1 to obtain hydration liquid;
mixing and dissolving DSPC and DSPE-PEG2000 in chloroform according to the mass ratio of 9: 1 to obtain a mixed solution;
(2) evaporating chloroform in the mixed solution through rotary evaporation to form a membrane to obtain a foaming body;
(3) adding a hydration liquid into the foaming body, and fully dissolving the foaming body by using ultrasound and a water bath at 65 ℃ until the solution becomes clear, and dissolving the foaming body in the hydration liquid to prepare a micro-bubble liquid; the content of the foaming body in the micro-bubble liquid is 3 mg/mL;
(4) and (3) sub-packaging the microbubble liquid into a penicillin bottle, and then replacing gas above the microbubble liquid in the penicillin bottle with perfluoropropane to prepare the microbubble composition. The ratio of the volume of the micro bubble liquid in the penicillin bottle to the volume of the perfluoropropane is 1: 4, and the pressure inside the penicillin bottle is balanced with the pressure outside the penicillin bottle.
Wherein, in the step (1), the mass ratio of the DSPC to the DSPE-PEG2000 in the mixed solution can be (8-10) to 1, and the volume ratio of the phosphate buffer saline solution, the glycerol and the propylene glycol in the hydration solution can be 8 to (0.9-1.1) to 1; in the step (3), the content of the foaming body in the micro-bubble liquid can be (2.5-3.5) mg/mL; in the step (4), the pressure inside the penicillin bottle can be controlled to be greater than or equal to the pressure outside the penicillin bottle.
The microbubble composition was stored in a refrigerator at 4 ℃. Before use, the solution filled in the penicillin bottle is horizontally vibrated by an oscillator to obtain the solution for forming the microbubbles. The microbubble formed from the microbubble composition of the present example has a structure of perfluoropropane, which is a lipid encapsulated inert gas, and the microscopic form thereof is shown in FIG. 1 (scale: 10 μm). The particle size distribution and concentration of microbubbles were measured using a Coulter counter Multisizer 4 using a small-bore tube of 20 μm in particle size, a range of 0.4 to 12 μm, and an average particle size of 1.179 μm, 90% of which were < 1.81 μm, and a microbubble concentration of 5.721X 109particle/mL, particle size distribution is shown in FIG. 2.
The medicine for opening the blood brain barrier in combination with ultrasound and treating amyotrophic lateral sclerosis consists of a microbubble composition and edaravone, wherein the ratio of the mass of edaravone to the volume of the microbubble composition is 75 mg: 1 mL. In other embodiments, the ratio of the volume of the microbubble composition to the mass of edaravone can also be (70-80) mg: 1 mL.
Four, ultrasonic therapy
The mouse is fixedly anesthetized on an ultrasonic platform, an ultrasonic probe is positioned on the motor cortex of the mouse, and after the microbubble composition is injected into the tail vein of the mouse, the ultrasonic is directionally applied to the cerebral motor cortex on two sides of the mouse. Wherein the ultrasonic parameters are as follows: the center frequency is 0.970MHz, the sound pressure is 0.63MPa, the repetition frequency is 1Hz, the irradiation duration is 60s, and the duty ratio is 10%.
Fluorescence imaging
3C 57 mice were taken, after one-time ultrasound treatment, evans blue was injected through the tail vein as a tracer, heart perfusion was performed 2 hours later using 4% paraformaldehyde, brains were taken, photographed under a light microscope, and fluorescence imaging was performed with a small animal fluorescence imager, as shown in FIG. 3.
Sixth, detecting edaravone content
10 SPF-grade C57BL/6J mice are taken, weighed to be 20-22 g, and divided into two groups after random numbering, wherein the two groups are a pure tail vein injection administration group (Eda group) and an ultrasonic combined microbubble administration group (FUS/MB + Eda group), and the administration dose is 5 mg/kg. Blood and mouse motor cortex areas are taken after administration for 5min, and the concentration of edaravone in serum and tissues is quantitatively determined by a liquid chromatography-mass spectrometry method, wherein the internal standard substance is phenacetin.
Seven, ultrasonic combined microbubble edaravone delivery brain treatment
50 13-week-old SOD1 female mice were randomly divided into five groups, namely, a model group (ALS group, n ═ 10), a simple ultrasound group (FUS group, n ═ 10), an ultrasound microbubble group (FUS/MB group, n ═ 10), a tail vein injection group (Eda group, n ═ 10), and an ultrasound microbubble administration group (FUS/MB + Eda), and 10 WT mice were simultaneously used as negative controls (WT group).
Mice were initially treated twice weekly at 13 weeks of age, with two to three days intervals between each dose for six weeks. The FUS/MB + Eda groups were administered as follows: the mouse is fixedly anesthetized on an ultrasonic platform, an ultrasonic probe is positioned on the motor cortex of the mouse, and after the mixed solution of the microbubble composition and the edaravone is injected into the tail vein of the mouse, ultrasonic waves are directionally applied to the motor cortex of the brain on two sides of the mouse. Wherein the ultrasonic parameters are as follows: the central frequency is 0.970MHz, the sound pressure is 0.63MPa, the repetition frequency is 1Hz, the irradiation duration is 60s, and the duty ratio is 10%; the injection amount of edaravone was 15mg/kg and the injection amount of the microbubble composition was 0.2. mu.L/g relative to the body weight of the mouse. The intravenous injections of other groups were replaced with saline (ALS group, FUS group), microbubbles (FUS/MB group) or edaravone injection (Eda group), while the ALS group and Eda group were not irradiated with ultrasound.
Eighthly, behavioural detection
The mice were subjected to one behavioral test before and four weeks after 13 weeks of treatment.
And (3) testing the grip strength: and (3) placing the mouse on the grabbing claw holding force test grid, pulling the tail of the mouse to enable four limbs of the mouse to tightly grab the test grid until the four limbs of the mouse are loosened, and recording the tension data on the tester. Maximum values were taken in triplicate for each mouse.
And (3) rotating a rod for testing: the mice are trained on a bar rotating instrument for 3min in 50s from 0r/min to 25r/min, and the training time is 10min for three times. During the test, the mouse falls off the rod due to the movement dysfunction on the rod rotating instrument accelerated from 0r/min to 25r/min within 50s, the computer automatically records the falling time, and the counting of the falling time is finished within 5min, and the counting is recorded for 5 min.
Nine, neuropathological testing
The brain tissue and spinal cord were subjected to paraffin-embedded section and hematoxylin-eosin (HE) staining, and the number of neurons was counted.
Ten, statistical processing
Statistical analysis was performed using software GraphPad Prism to measure data
Indicated, the group comparisons were analyzed using Tukey's multiple comparison test, and pairwise comparisons were performed using t-test.
Eleventh, experimental results
1. Blood brain barrier result of mice opened by ultrasound combined with microbubbles
After the mouse BBB is opened by ultrasound in combination with microbubbles to deliver evans blue into the brain, it can be seen from the light microscope picture (fig. 3) of the brain that the ultrasound microbubbles successfully opened the blood brain barrier and allowed evans blue to enter the cortical region. In fluorescence imaging, evans blue can enter deep into the brain after opening the blood brain barrier, covering the cortical region (fig. 3). This indicates that the ultrasound microbubbles opened the entry of substances in the blood-brain barrier into the brain tissue.
2. Edaravone content detection result
The establishment result of the method for determining the content of edaravone in the sample is as follows: the linear relation of the concentration of the edaravone is good within the range of 20-10000 ng/mL, wherein the ratio Y of the peak area of the edaravone to the peak area of the phenacetin is taken as the ordinate,the concentration C of edaravone is abscissa, and a standard curve Y is obtained by regression with a weighting coefficient (1/X2) — 0.00126604+0.00032C, R2＝0.9968。
Fig. 4 shows the percent brain drug/blood ratio administered, with the mean value of group Eda being 0.1846 ± 0.01776(N ═ 5) and the mean value of group FUS/MB + Eda being 0.6163 ± 0.1316(N ═ 5). Compared with the simple tail vein injection, the brain drug/blood drug ratio percentage of the ultrasound combined microbubble for opening the blood brain barrier and delivering the edaravone into the brain is improved by 0.43 percent (p is less than 0.05). The result shows that the brain bioavailability of edaravone is improved by the ultrasound and microbubble combined blood brain barrier opening technology.
3. Behavioral results
Prior to treatment of mice at 13 weeks of age, the paw grip of the SOD1 group mice was 129.9 ± 2.020mg (N ═ 49), which was a 43% decrease (p < 0.0001) relative to WT mice (228.2 ± 8.381mg, N ═ 10), as shown in figure 5. Therefore, the SOD1 group mice had significantly reduced paw grip relative to the WT group mice. Groups of SOD1 mice were randomized into groups for 4 weeks of treatment before testing for paw grip. To eliminate the basal differences before treatment for each experimental mouse, the data processing used relative growth ratio statistics ((4 weeks post-treatment paw grip-pre-treatment paw grip)/pre-treatment paw grip) as shown in fig. 6. The relative increase ratio of the claw holding power of the mice in the FUS/MB + Eda group is-24.13 (N is 10), and the claw holding power of the mice in the ALS group is increased by 10.57 percent (p is less than 0.05) compared with the mice in the ALS group (34.7, N is 10). The improvement of 9.11% (p < 0.05) was observed between FUS/MB + Eda and Eda (-33.24, N ═ 10).
FIG. 7 shows the results of a mouse rotarod behavioural test. The retention time of mice in the SOD1 group on the rotarod before 13-week-old treatment was 263.4 ± 7.331s (N ═ 50, decreased by 36.61 ± 16.34s (p < 0.05) compared to WT mice (300.0 ± 0.0s, N ═ 10), so that the retention time of mice in the SOD1 group on the rotarod was 234.0 ± 20.75s (N ═ 10) compared to WT mice, after 4 weeks of treatment, the retention time of mice in the FUS/MB + Eda group on the rotarod was 234.0 ± 20.75s (N ═ 10), the exercise time was increased by 103.9 ± 33.03s (p < 0.01) compared to Eda group (148.8 ± 18.63s, N ═ 10) and the exercise time was increased by 85.20 ± 27.88s (p < 0.05).
These results indicate that ultrasound in combination with microbubble delivery of edaravone into the brain more effectively delayed disease progression in SOD1 mice than the single tail vein administration, at the same dose of treatment.
4. The result of neuronal degeneration
Morphological changes of anterior horn motor neurons of the lumbar cord after 6 weeks of treatment of the WT group, the model group (ALS group), the edaravone treatment group (Eda group) and the ultrasound-combined microbubble delivery edaravone treatment group (FUS/MB + Eda group) mice (5 mice per group) were observed by HE staining and the motor neurons were counted. HE staining results are shown in fig. 8, a: WT group, B: ALS group, C: eds group, D: FUS/MB + Eda set), scale: 20 μm; the ALS group (B picture) mice have obviously reduced numbers of anterior lumbar-medullary motor neurons and cell atrophy compared with the WT group (A picture), and quantitative analysis shows that the number of neurons in the ALS group is reduced by 69.0%, the number of neurons in the Eda group is reduced by 59.1%, and the number of neurons in the FUS/MB + Eda group is reduced by 50.7% compared with the WT group. The results show that the ultrasonic combined microbubble-mediated edaravone delivery into the brain can improve the morphological lesion of the lumbar anterior cord neuron of the SOD1 mouse, and has more effect compared with a simple administration group. Motor neuron counts are shown in fig. 9, where: p < 0.01, x: p is less than 0.0001.
According to the experimental results, under the condition of the same dose of treatment, the method disclosed by the invention is applied, and edaravone is delivered to the brain through ultrasound and micro-bubble, so that the disease progress of the SOD1 mouse is more effectively delayed compared with a drug delivery mode of single tail vein injection.
Twelve, influence of drug combination consisting of edaravone and microbubble compositions with different proportions on blood brain barrier of mice
Mice were initially treated twice weekly at 13 weeks of age, with two to three days intervals between each dose for six weeks.
The administration mode is as follows: the mouse is fixedly anesthetized on an ultrasonic platform, an ultrasonic probe is positioned on the motor cortex of the mouse, the mixed solution of the microbubble composition and the edaravone is injected into the tail vein of the mouse (with evans blue added as a tracer), and then ultrasonic is directionally applied to the motor cortex of the brain on two sides of the mouse. The ultrasonic parameters are as follows: the center frequency is 0.970MHz, the sound pressure is 0.63MPa, the repetition frequency is 1Hz, the irradiation duration is 60s, and the duty ratio is 10%.
The injection amounts for comparative test 1 were: relative to the body weight of the mouse, the injection amount of the edaravone is 15mg/kg, and the injection amount of the microbubble composition is 0.1 mu L/g; that is, the ratio of the mass of edaravone to the volume of the microbubble composition was 150 mg: 1 mL.
The injection amounts for comparative experiment 2 were: relative to the body weight of the mouse, the injection amount of the edaravone is 15mg/kg, and the injection amount of the microbubble composition is 0.3 mu L/g; that is, the ratio of the mass of edaravone to the volume of the microbubble composition was 50 mg: 1 mL.
After the evans blue is delivered into the brain by the ultrasound-combined microbubble open mouse BBB, as can be seen from the light microscope picture of the taken brain, in the comparative experiment 1, the blood brain barrier opening of the mouse is smaller because the ratio of the mass of the injected edaravone to the volume of the microbubble composition is 150 mg: 1mL, as shown in FIG. 10; in contrast, in comparative experiment 2, the blood-brain barrier of the mice was excessively opened because the ratio of the mass of the injected edaravone to the volume of the microbubble composition was 50 mg: 1mL, as shown in fig. 11. The too small or too large opening degree of the comparative test 1 and the comparative test 2 to the blood brain barrier of the mouse is not beneficial to improving the treatment effect on ALS.
The technical contents of the present invention are further illustrated by the examples, so as to facilitate the understanding of the reader, but the embodiments of the present invention are not limited thereto, and any technical extension or re-creation based on the present invention is protected by the present invention.
Claims (10)
1. The medicine for treating amyotrophic lateral sclerosis is characterized by consisting of a microbubble composition and edaravone, wherein the microbubble composition consists of microbubble liquid and perfluoropropane, and the microbubble liquid consists of a foaming body and a hydration liquid;
the foaming body consists of DSPC and DSPE-PEG2000 according to the mass ratio of (8-10) to 1;
the hydration liquid is formed by mixing phosphate buffer saline solution, glycerol and propylene glycol according to the volume ratio of 8: 0.9-1.1: 1;
the content of the foam in the micro-bubble liquid is (2.5-3.5) mg/mL.
2. The drug for treating amyotrophic lateral sclerosis according to claim 1, wherein the microbubble composition is horizontally oscillated to form microbubbles with particle size of 0.4-12 μm, more than 90% of the microbubbles have particle size of less than 1.81 μm, and the concentration of the microbubbles is greater than or equal to 5.721 x 109one/mL.
3. The medicament for treating amyotrophic lateral sclerosis according to claim 1, wherein the foam is composed of DSPC and DSPE-PEG2000 in a mass ratio of 9: 1; the hydration liquid is formed by mixing phosphate buffer salt solution, glycerol and propylene glycol according to the volume ratio of 8: 1; the content of the foam in the microbubble liquid was 3 mg/mL.
4. The medicament for treating amyotrophic lateral sclerosis according to any one of claims 1 to 3, wherein the ratio of the volume of the microvesicle liquid to the volume of perfluoropropane is 1: 4.
5. The medicament for treating amyotrophic lateral sclerosis according to claim 4, wherein the microbubble composition is contained in a sealed container, and a pressure inside the sealed container is greater than or equal to a pressure outside the sealed container.
6. The medicament for treating amyotrophic lateral sclerosis according to claim 5, wherein the ratio of the mass of edaravone to the volume of microbubble composition is (70-80) mg: 1 mL.
7. The medicament for treating amyotrophic lateral sclerosis according to claim 6, wherein the ratio of the mass of edaravone to the volume of microbubble composition is 75 mg: 1 mL.
8. The medicament for treating amyotrophic lateral sclerosis according to claim 1, characterized by being prepared by:
s1, uniformly mixing phosphate buffer salt solution, glycerol and propylene glycol to obtain a hydration solution;
dissolving DSPC and DSPE-PEG2000 in chloroform to obtain a mixed solution;
s2, evaporating the chloroform in the mixed solution to form a foaming body;
s4, adding a hydration liquid into the foaming body to dissolve the foaming body into the hydration liquid to prepare micro-bubble liquid;
s5, filling the micro-bubble liquid into a sealed container, and then replacing gas above the micro-bubble liquid with perfluoropropane to prepare a micro-bubble composition;
s6, the microbubble composition and the edaravone are combined to form the medicine.
9. The medicament for treating amyotrophic lateral sclerosis according to claim 8, wherein the ratio of the volume of the microbubble liquid to the volume of the perfluoropropane is 1: 4, and the pressure inside the sealed container is greater than or equal to the pressure outside the sealed container.
10. A method for improving the opening effect of the blood-brain barrier, characterized in that after the intravenous injection of the medicament according to any one of claims 1 to 9, ultrasound is directionally applied to the bilateral cerebral motor cortex, and the ultrasound parameters are as follows: the center frequency is 0.970MHz, the sound pressure is 0.63MPa, the repetition frequency is 1Hz, the irradiation duration is 60s, and the duty ratio is 10%.
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