Oily butylphthalide gel for treating traumatic brain injury as well as preparation method and application of oily butylphthalide gel

Abstract

The invention relates to oily butylphthalide gel for treating traumatic brain injury as well as a preparation method and application of the oily butylphthalide gel. The preparation method of the oily butylphthalide gel comprises the following steps: mixing a proper amount of ethyl isobutyl sucrose ester (SAIB) and butylphthalide, adding a small amount of pure water, and violently shaking to obtain the oily butylphthalide gel. Experimental results show that the oily butylphthalide gel prepared by the method has modulus which is very matched with that of brain tissues and is beneficial to tissue regeneration. Therefore, the oily butylphthalide gel disclosed by the invention has an excellent effect of treating traumatic encephalopathy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of drug carriers, and particularly relates to an oily butylphthalide gel for treating traumatic brain injury and a preparation method and use thereof. BACKGROUND

[0002] Traumatic brain injury (TBI) is a traumatic structural injury or physiological damage to the brain caused by external force, and is one of the most common brain diseases in neurosurgery and emergency. Globally, there are approximately 5-6 million TBI patients per year. Among them, the largest cause of TBI in developed countries is falling (53%), followed by road traffic accidents (26%), while in developing countries, the largest cause of TBI is road traffic accidents (57%), followed by falling (20%). In addition, various violence including wars, regional conflicts, brawls, etc. are also important causes of TBI.

[0003] In addition to the primary injury to the brain caused by external force, TBI patients usually also suffer from secondary injury. After the occurrence of primary injury, various pathological changes such as oxidative stress and inflammatory response occur in the injured tissue. If these early pathological changes are not treated in time, these changes will lead to secondary brain injury, which plays a more important role in the development of TBI than the primary brain injury. Reactive astrocytes and activated microglia recruited to the injured tissue will further enhance neuroinflammation, release reactive oxygen species (ROS), inhibit the integration of newly formed neurons, and prevent axon development. In addition, the accumulation of ROS in the brain trauma microenvironment creates a toxic environment for adjacent neurons and releases a large amount of inflammatory factors, which can induce apoptosis and necrosis of neural cells through activated microglia, thereby further leading to neural loss. These secondary injuries can further cause impaired neural function, thereby triggering motor disorders, decreased cognitive ability, emotional instability, speech disorders, and behavioral changes in patients. However, due to the complex pathology, lack of clinical drugs, and difficulty in drug delivery to the brain and targeting, so far there is no recognized successful TBI treatment method in clinical practice.

[0004] While a variety of drugs have been reported in the literature to be useful in the rehabilitation of TBI, how to effectively deliver the drugs to the brain is also a problem to be solved. With the gradual recovery of the blood-brain barrier after TBI, it is more and more difficult for traditional intravenous injection therapy to deliver drugs to the brain. Therefore, multiple doses are required to achieve therapeutic effect, which often leads to various side effects such as liver toxicity, intestinal dysbiosis, and gastrointestinal discomfort. Part of the mild TBI patients and more than 90% of the moderate and severe TBI need to receive surgical treatment to remove blood clots, broken bones and foreign bodies. After surgery, more space is opened in the brain to form a fillable cavity. Currently, only limited postoperative treatments such as hemostatic sponges are given in the clinic. Therefore, by using the cavity formed after TBI surgery and using hydrogel carriers to load drugs, not only can the drugs be directly delivered to the disease site, but also the sustained release characteristics of hydrogels can achieve long-term release of drugs to continuously promote the rehabilitation after TBI. The injectable gel in liquid form can form a gel in situ and continuously release drugs in situ, which is very suitable for the treatment of brain diseases, and its injectability ensures that the drugs directly enter the target lesion. In addition, it can also be endowed with intelligent release behavior, such as ROS-responsive release, to significantly improve the bioavailability and drug efficacy of the drugs.

[0005] Dlpropylphrine is an active ingredient extracted from celery seeds, also known as apigenin, which is very effective in improving the neurological impairment of patients with acute ischemic stroke. Dlpropylphrine can protect the cerebral blood vessels and blood-brain barrier, reduce the oxidative stress of injured brain tissue, protect mitochondria, improve brain metabolism and thus block the pathological process of secondary brain injury. In addition, compared with other drugs commonly used in the clinic for the treatment of TBI, dlpropylphrine has fewer adverse reactions. Therefore, it is of great clinical significance and commercial value to develop a suitable drug delivery system for dlpropylphrine for the treatment of TBI to further increase its efficacy and reduce its adverse reactions.

[0006] In view of this, the present inventors propose a new oily dlpropylphrine gel delivery system for the treatment of traumatic brain injury. SUMMARY

[0007] In view of the deficiencies of the prior art, the present application provides an oily dlpropylphrine gel for the treatment of traumatic brain injury, as well as a preparation method and use thereof.

[0008] To achieve the above-mentioned purpose, the technical solution adopted by the present application is as follows:

[0009] In a first aspect, the present application provides a preparation method of an oily dlpropylphrine gel for the treatment of traumatic brain injury, comprising the following steps:

[0010] Take the appropriate amount of ethyl isobutyl sucrose ester (SAIB) and butylphthalide mixture, and then add a small amount of pure water, and shake vigorously, and the oil butylphthalide gel can be obtained.

[0011] Ethyl isobutyl sucrose ester (SAIB): the mechanism of SAIB and water mixed to form a gel mainly depends on the hydrophobic-hydrophilic balance of its molecular structure and intermolecular interaction. The hydrophobic acetyl and isobutyryl of SAIB promotes the aggregation of molecules in water through hydrophobic interaction, forming a hydrophobic micro zone, while a small amount of residual hydroxyl forms a hydrogen bond network with water, and the spatial steric hindrance and entanglement between molecules further stabilize the structure. When the critical concentration is reached, these aggregates are connected to each other to form a three-dimensional network, which encapsulates water, and finally forms a gel with thermal reversibility and shear thinning properties. The photo of SAIB and the gel formed by SAIB and water is shown in Figure 1 .

[0012] As an optional way, in the above preparation method, the volume ratio of SAIB to butylphthalide is (1-10): 1.

[0013] As an optional way, in the above preparation method, the volume ratio of SAIB to butylphthalide is 5:1.

[0014] As an optional way, in the above preparation method, the volume ratio of butylphthalide to pure water is (2-5): 1.

[0015] Preferably, the preparation method of the oil butylphthalide gel is as follows: take 1 mL of SAIB and 0.2 mL of butylphthalide, and then add 40-100 μL of pure water, and shake vigorously, and the oil butylphthalide gel can be obtained.

[0016] For example, the preparation method of the oil butylphthalide gel is as follows: take 1 mL of SAIB and 0.2 mL of butylphthalide, and then add 50 μL of pure water, and shake vigorously, and the oil butylphthalide gel can be obtained.

[0017] Alternatively, for example, the preparation method of the oil butylphthalide gel is as follows: take 1 mL of SAIB and 0.2 mL of butylphthalide, and then add 100 μL of pure water, and shake vigorously, and the oil butylphthalide gel can be obtained.

[0018] As an optional way, in the above preparation method, the butylphthalide is an oily liquid with a celery aroma.

[0019] In a second aspect, the present application provides an oil butylphthalide gel prepared by the preparation method of the first aspect described above, which has a modulus that is very matched with brain tissue, and is beneficial to tissue regeneration.

[0020] In a third aspect, the present application provides the use of the oily butylphthalide gel of the first aspect above in the manufacture of a product for the treatment of traumatic brain injury.

[0021] Alternatively, in the use above, the product is an injectable gel filler.

[0022] Compared with the prior art, the present application has the following advantages and positive effects:

[0023] In the preparation method of the present application, the reaction conditions are mild, the steps are simple, and the preparation efficiency is high. Experimental results show that the oily butylphthalide gel prepared by the method of the present application has a modulus that is very matched with brain tissue, which is beneficial to tissue regeneration. Therefore, the oily butylphthalide gel of the present application has excellent effect on the treatment of traumatic brain disease. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Photos showing SAIB (A) and its gel formation with water (B).

[0025] Figure 2 Modulus of the oily butylphthalide gel with different water contents.

[0026] Figure 3 Photo of preparation of TBI animal model by Feeney free fall method.

[0027] Figure 4 Rotarod test results of the oily butylphthalide gel carrier.

[0028] Figure 5 Balance beam test results of the oily butylphthalide gel carrier.

[0029] Figure 6 Effects of the oily butylphthalide gel carrier on the results of in vivo characterization of neural plasticity. DETAILED DESCRIPTION

[0030] The present application will be further described below with reference to specific examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the scope of the present application.

[0031] Unless otherwise specified, the specific techniques or conditions in the examples are carried out according to the techniques or conditions described in the literature in the art, or according to the product instructions. Unless otherwise specified, the reagents or instruments used are conventional products that can be purchased through regular channels.

[0032] Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the test materials used in the following examples are commercially available products.

[0033] Preparation Example:

[0034] 1. Materials

[0035] Butylphthalide (an oily liquid with a celery aroma, Hubei Xinkang Pharmaceutical Chemical Co., Ltd.); Ethyl isobutyl sucrose ester (SAIB) (Zancheng (Tianjin) Technology Co., Ltd.).

[0036] 2. Preparation method of oily butylphthalide gel

[0037] The preparation method of the oily butylphthalide gel is as follows: 1 mL of SAIB and 0.2 mL of butylphthalide are mixed, 50 μL or 100 μL of pure water is added, and the mixture is shaken vigorously to obtain the oily butylphthalide gel.

[0038] The oily butylphthalide gels used in the following effect examples are all prepared according to the preparation method in the “Preparation Examples” section.

[0039] Effect Examples:

[0040] Effect Example 1: Performance research results of the oily lomer gel of the present application

[0041] 1. Experimental method

[0042] The rheological properties of the oily butylphthalide gel were characterized using a rheometer (MCR 301 Anton Paar, Ashland, VA, US). The instrument was preheated, and a parallel plate rotor (e.g., 25 mm) was selected for zero-point calibration. The test temperature (e.g., 25°C) was set, and the temperature was allowed to stabilize. The upper plate was raised, and the gel sample was evenly spread on the center of the lower plate to avoid air bubbles. The upper plate was slowly lowered to the set gap (usually 1 mm), and the excess sample was gently removed with a spatula, and the temperature was allowed to equilibrate for 5-15 min. The strain control mode (0.1%) was selected, and the test was started. After the test, the rotor was thoroughly cleaned.

[0043] 2. Experimental results

[0044] Figure 2 The moduli of the oily butylphthalide gels with different water contents are shown. At a water addition amount of 50 μL, the gel loss modulus (G”) is slightly greater than the storage modulus (G’), and the gel still has the viscoelastic characteristics of a slightly liquid. When the water addition amount reaches 100 μL, the G’>G” of the gel, indicating that the gel has the viscoelastic characteristics of a solid gel. In addition, the experimental results show that the oily butylphthalide gel has a modulus that is very matched to brain tissue, which is conducive to tissue regeneration. Effect Example 2: Pharmacodynamic research results of the oily lomer gel of the present application in a TBI animal model Figure 3

[0045] 1. Experimental method

[0046] 1.1 Animals

[0047] All in vivo experiments were approved by the Animal Ethics Committee of Beijing Rehabilitation Hospital, Capital Medical University. All animal care, housing, surgery, and anesthesia procedures were performed in accordance with the Regulations for the Administration of Laboratory Animals of China. Male C57BL / 6j mice (8 weeks old, 24.0 ± 1.5 g) were used in the experiments. Laboratory chow and water were provided for 7 days to acclimate the animals to the environment.

[0048] 1.2 Establishment of a mouse model of moderate traumatic brain injury (TBI)

[0049] Mice were anesthetized with isoflurane (2% induction, 1.5% maintenance), shaved, and the scalp was disinfected. Then, the mice were fixed on a stereotaxic apparatus (Shenzhen Ruivode Technology Co., Ltd.), a midline incision was made along the head-tail axis to separate the skull from the skin, and the skull was exposed. The TBI animal model was established using the typical Feeney free-fall method. A 1.5-cm incision was made on the midline of the scalp, the skull was opened at 2 mm to the right of the midline and 3 mm behind the bregma, and a 4-mm-diameter skull was carefully removed. The impactor was placed perpendicular to the dura mater. When the impactor contacted the dura mater, a 40-gram weight was dropped from a height of 7.5 cm to cause moderate brain injury. As shown in Fig. 1B. After debridement and hemostasis, the TBI mice were randomly divided into 5 groups of 6 mice each, including Sham (sham operation group), TBI (brain injury group), TBI+Blank (brain injury treated with blank SAIB gel), TBI+aq (brain injury treated with butylphthalide), and TBI+gel (brain injury treated with butylphthalide-SAIB). The sham group was not hit and the scalp was sutured directly; the TBI group was not treated with any material after the hit. After debridement and hemostasis, 10 μL of blank gel or butylphthalide-containing gel or the corresponding amount of butylphthalide was injected into the brain injury model cavity using a 10-μL microsyringe for 1 min. Finally, the mouse head skin was sutured and placed on a heating pad (37°C) for 10 min. When the mice recovered from anesthesia, they were placed in a new cage and food and water were supplemented as needed. Figure 4

[0050] 1.3 Behavioral assessment of animals

[0051] Rotarod test: Mice were trained on a rotarod (Panlab Rotarod LE8505) at a speed of 10 revolutions per minute for 3 days before the previous test. During the experiment, the speed of the rotating rod was uniformly accelerated from 4 to 40 revolutions in 5 min, and the time for the mouse to fall off the rotating shaft was recorded.

[0052] Balance beam test: Motor coordination was quantified by counting the number of times the right hind paw slipped off the balance beam (about 80 cm long, about 1.5 mm wide, 100 cm from the ground) during the experiment.

[0053] ​The above experiments were trained for 3 days before formal testing, and each mouse was repeated three times.

[0054] 1.4 In vivo characterization of neural plasticity

[0055] On day 21 after brain injury, mice were anesthetized with 1% sodium pentobarbital at a dose of 0.3 mL. After exposing the heart, about 50 mL of 0.9% saline was immediately perfused from the left ventricle, followed by about 50 mL of 4% PFA. Brain tissue was isolated and stored in PFA (4%) for fixation. Brain tissue was collected from the edge area of the cerebral cortex injury, and IF staining was used to detect the expression of Vglutl and PSD95 to study synaptic plasticity in vivo.

[0056] The brain tissue was embedded in paraffin, and 4 pm sections were cut with a microtome (Leica RM2235, Germany). Immunofluorescence staining was performed using mouse anti-PSD95 (ab2723, Abeam, UK) and rabbit anti-vglutl (GTX133148, GeneTex, USA). The sections were scanned for fluorescence using a digital section scanner (KF-PRO-020, KFBIO, Ningbo Jiangfeng Biological Information Co., Ltd.). Finally, the sections were scanned for fluorescence using a digital section scanner (KF-PRO-020, KFBIO, Ningbo Jiangfeng Biological Information Co., Ltd.).

[0057] 1.5 Data representation method

[0058] Statistical analysis was performed using SPSS software (version 20; IBM, Armonk, NY). The results are expressed as mean ± standard deviation (SD). The differences between the two groups were tested using t-test. All analyses were considered statistically significant at p<0.05, where *p<0.05, **p<0.01, ***p<0.001.

[0059] 2. Experimental results

[0060] As Figure 5 and Figure 6As shown, the rotisserie and balance beam experiments demonstrate the effect of oily butylphthalide gel in promoting the recovery of motor function in TBI mice. Because they did not receive TBI modeling, the fall time from the rotisserie in the Sham group remained consistently high. However, after TBI modeling, the fall time in all groups decreased sharply, and then gradually recovered over time. In the three treatment groups, the fall time in the TBI+gel group was consistently significantly higher than the other two groups (TBI+blank, TBI+aq), which fully demonstrates the efficacy of oily butylphthalide gel in promoting the recovery of motor function in TBI mice. The balance beam experiment showed a similar trend: the number of hindlimb falls in the Sham group remained consistently low during the test; the number of falls in the other groups increased rapidly after TBI modeling, and then gradually recovered over time. However, at each test time point, the number of falls in the TBI+gel group was significantly less than the other two groups, further demonstrating the efficacy of oily butylphthalide gel in treating TBI mice. It is worth noting that in both behavioral tests, the efficacy of butylphthalide solution treatment (TBI+aq) was significantly lower than that of oil-based butylphthalide gel treatment (TBI+gel), which also illustrates the advantage of oil-based butylphthalide gel treatment over butylphthalide solution treatment.

[0061] PSD95 and Vglut-1 are markers of synaptic plasticity, and their co-localization characterizes neural plasticity. For example... ​ As shown, the immunofluorescence results are consistent with the behavioral results, indicating a significant decrease in motor function and a marked reduction in synaptic connections in mice after TBI. After treatment with oily butylphthalide gel, both motor function and synaptic connections in the mice significantly recovered, with the order of synaptic connection strength being sham > TBI+gel > TBI+aq > TBI+blank > TBI.

[0062] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method of preparing an oily guguglll gel of butylphthalide for treating traumatic brain injury, characterized in that: The preparation method comprises the following steps: taking appropriate amount of ethyl isobutyl sucrose ester (SAIB) and butylphthalide, mixing, adding a small amount of pure water, and shaking intensively to obtain the oily butylphthalide gel.

2. The method of claim 1, wherein: The volume ratio of the SAIB to the butylphthalide is (1-10):

1.

3. The method of claim 1, wherein: The volume ratio of the SAIB to the butylphthalide is 5:

1.

4. The method of claim 1, wherein: The volume ratio of the butylphthalide to the pure water is (2-5):

1.

5. The method of claim 1, wherein: The butylphthalide is an oily liquid with celery fragrance.

6. The oily butylphthalide gel prepared by the preparation method according to any one of claims 1 to 5, characterized in that: The oily butylphthalide gel has a modulus very matched with brain tissue, and is beneficial to tissue regeneration.

7. Use of the oily butylphthalide gel in claim 6 in the preparation of a product for treating traumatic brain injury.

8. Use according to claim 7, characterized in that: The product is an injectable gel filler.

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