CN111265519B

CN111265519B - Pharmaceutical composition for treating traumatic brain injury and preparation thereof

Info

Publication number: CN111265519B
Application number: CN202010220161.7A
Authority: CN
Inventors: 魏晓捷; 许信龙; 陈再丰; 邬芬赞; 张宏宇
Original assignee: Cixi People's Hospital Medical And Health Group Cixi People's Hospital
Current assignee: Cixi People's Hospital Medical And Health Group Cixi People's Hospital
Priority date: 2020-03-25
Filing date: 2020-03-25
Publication date: 2021-02-19
Anticipated expiration: 2040-03-25
Also published as: CN111265519A

Abstract

The invention provides a pharmaceutical composition for treating traumatic brain injury and a preparation thereof, wherein the pharmaceutical composition comprises edaravone, isoxsuprine and a pharmaceutically acceptable carrier. The pharmaceutical composition can improve the nerve function after brain trauma, relieve the encephaledema degree after brain trauma, inhibit nerve cell apoptosis and reduce the central inflammatory reaction mediated by microglia.

Description

Pharmaceutical composition for treating traumatic brain injury and preparation thereof

Technical Field

The invention belongs to the technical field of medicines, relates to a medicine for treating brain injury, and particularly relates to a pharmaceutical composition for treating traumatic brain injury and a preparation thereof.

Background

Traumatic Brain Injury (TBI), also known as intracranial injury, is a common disease in neurosurgery and is one of the well-known frequently-occurring and difficult-to-heal injuries. A large number of studies have confirmed that post-TBI brain dysfunction is not only due to the effects of primary injury such as initial mechanical forces, but also is largely associated with the occurrence of complex neuronal "secondary hits" after injury, i.e., secondary neuronal injury, whose mechanisms mainly include calcium overload, excitatory amino acid toxicity effects, mitochondrial dysfunction, oxidative stress, apoptosis, and inflammatory responses.

Early after TBI occurs, there is a massive proinflammatory cytokine release and immune cell activation. Activation of immune cells, in turn, leads to the release of free radicals and more inflammatory mediators. Regardless of the factor by which this inflammatory cascade is initiated, inhibition of the inflammatory response may reduce cerebral edema, and reduce neuronal apoptosis or gliosis. Inflammatory responses are the most prominent pathological process of secondary brain injury, in which inflammatory factors play an important role in inflammatory responses after brain trauma, and many inflammatory factors are highly expressed after brain injury and are considered to be closely related to the degree of brain injury. TNF-alpha is an important proinflammatory cytokine released earlier, can activate cytokine cascade reaction in the inflammatory reaction process, induces the synthesis of IL and secondary inflammatory mediators, and can cause cerebral edema and blood brain barrier destruction to aggravate brain tissue damage. IL-6, IL-1 alpha, IL-1 beta are also an important inflammatory cytokine in the inflammatory response chain of brain injury; IL-10, as an anti-inflammatory factor, inhibits inflammatory cell activation, migration, adhesion, reduces the synthesis and release of various inflammatory cytokines, and reduces excessive neuronal apoptosis. After TBI, inflammatory cells such as macrophages and the like can be activated and oxygen free radicals can be activated, so that cytokine in brain is synthesized and released, and the immune balance of the body is disturbed. Therefore, the fact that the brain injury can increase the expression of the proinflammatory factors and correspondingly increase the secretion of the anti-inflammatory factors is suggested, because the wound causes the inflammatory reaction and is accompanied with the compensatory anti-inflammatory reaction, the compensatory anti-inflammatory factors are generated to resist the inflammatory factors, and the damage to the self tissues caused by the inflammatory reaction is favorably improved.

Traumatic subarachnoid hemorrhage is considered to be an important factor in cerebral vasospasm after TBI, with middle cerebral artery spasm being the most common. Traumatic subarachnoid hemorrhage is mainly caused by cerebral surface vascular injury or intracranial bridge vein injury caused by TBI, is common in TBI patients and is also an important reason for aggravating secondary TBI. The main complication of traumatic subarachnoid hemorrhage is cerebral vasospasm, which is also one of the important indicators for judging the prognosis of severe TBI. Independent risk factors for post-TBI vasospasm include contusion and inflammatory response of the brain parenchyma. Activation of a wide range of mechanical injury and inflammatory pathways is thought to be a potential mechanism for vasospasm to occur following TBI. Pathophysiology and cellular events of early TBI play an important role in patient prognosis, including increased intracranial pressure, decreased cerebral blood flow, impaired blood-brain barrier due to cerebral ischemia, inflammation, hypoxia.

Edaravone (Edaravone) is a radical scavenger, and as a cerebral protective agent, it has been confirmed to have a therapeutic effect on various cerebral diseases such as cerebral ischemia reperfusion, parkinson disease, alzheimer disease, and the like. Existing research shows that in a TBI model, edaravone can improve neurobehavioral disorders of rats after TBI, reduce the degree of cerebral edema, inhibit the damage of a blood brain barrier, and reverse histopathological changes (experimental research on the brain protection effect of edaravone on rats with traumatic brain injury, Mabajun et al, traffic medicine, 2008, Vol 22, No 6). Further research shows that edaravone can regulate the apoptosis pathway of nerve cells after TBI and has a protective effect on TBI (edaravone influences the signal pathway of extracellular signal-regulated kinase 1/2 after heavy diffuse brain trauma in rats, China is critical illness emergency medicine, Vol. 22, No. 4 of 2010). Further studies showed that edaravone treatment can reduce oxidative stress induced by traumatic brain injury in mice after TBI (influence of edaravone on oxidative stress after traumatic brain injury in mice, journal of chinese emergency medicine, vol.28, No. 3, 2019). But no influence of edaravone on the expression of inflammatory factors and immune cell activation after TBI has been found yet.

Isoxaprine (Isoxsuprine) is a vasodilator, can help increase blood flow in multiple blood vessels throughout the body, and can be used for treating arteriosclerosis, Raynaud's disease, thromboangiitis obliterans, cerebrovascular and peripheral vasospastic diseases. However, the prior art has no description about the treatment effect of the TBI in traumatic brain injury.

Disclosure of Invention

The edaravone can obviously reduce the degree of cerebral edema after TBI, inhibit nerve cell apoptosis, improve nerve behaviors, reduce microglial cell-mediated central inflammatory reaction and reduce the expression of TNF-alpha, IL1 alpha, IL-1 beta and IL 6. The invention unexpectedly discovers that the pharmaceutical combination of the edaravone and the isochrysol can generate synergistic effect on preventing and/or treating Traumatic Brain Injury (TBI).

Accordingly, in a first aspect, the present invention provides a pharmaceutical composition for use in the treatment of traumatic brain injury, characterised in that the pharmaceutical composition comprises edaravone and isoxsuprine, and a pharmaceutically acceptable carrier.

The weight ratio of the edaravone to the isoxsuprine in the pharmaceutical composition is 1:5 to 5:1, and preferably the weight ratio of the edaravone to the isoxsuprine is 3: 1.

Furthermore, the pharmaceutical composition is used for improving the nerve function after the brain trauma after the traumatic brain injury, relieving the brain edema degree after the brain trauma, inhibiting the nerve cell apoptosis and reducing the central inflammatory reaction mediated by the microglia.

Further, the traumatic brain injury is selected from concussion, closed craniocerebral injury or open craniocerebral injury.

In a second aspect of the present invention, there is provided a formulation for use in the treatment of traumatic brain injury, wherein the formulation comprises a pharmaceutical composition according to the first aspect.

Further, the formulation is in the form of tablet, granule, capsule, suspension, oral liquid, injection, transdermal patch, or inhalant.

In a third aspect of the invention, the application of the pharmaceutical composition containing edaravone and isoxsuprine in preparing a medicament for preventing and/or treating traumatic brain injury is provided.

The pharmaceutical compositions and formulations thereof of the present invention may be used by any route capable of delivery to the brain. Examples of routes of administration include, but are not limited to, intravenous, intranasal, oral, topical, scalp or administration by inhalation.

In accordance with the pharmaceutical compositions of the present invention, for oral administration of the pharmaceutical compositions, a therapeutically effective amount of one or more of the compounds of the present invention is preferably mixed with a pharmaceutically acceptable carrier according to conventional quantitative pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral. In preparing the pharmaceutical compositions in oral dosage form, any of the usual pharmaceutical media may be employed. Thus, for liquid oral preparations such as suspensions, elixirs and solutions, suitable carriers and additives including water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like may be used. For solid oral preparations such as powders, tablets, capsules, and solid dosage forms such as suppositories, suitable carriers and additives include starches, sugar carriers such as glucose, mannitol, lactose and related carriers, diluents, granulating agents, lubricants, binders, disintegrating agents and the like may be used. Tablets or capsules may be enteric coated for sustained release by standard techniques, if desired. The bioavailability of the compounds in the patient can be significantly affected by the use of these dosage forms.

For parenteral formulations, the carrier will usually comprise sterile water or aqueous sodium chloride solution, and other ingredients such as dispersing aids may also be included. When sterile water is to be used or the sterility of sterile water is to be maintained, the compositions and carriers must also be sterilized. Can also be prepared as suspensions for injection, in which case suitable liquid carriers, suspending agents and the like can also be used.

The pharmaceutical compositions of the present invention may be administered in a single dose or in multiple doses. Wherein the single administration dose of the edaravone is in the range of 1-150mg, the single administration dose of the isochrysol is in the range of 1-50mg, and the preferred single administration dose of the edaravone is 30mg and the single administration dose of the isochrysol is 10 mg.

Detailed Description

The following non-limiting examples are provided to further illustrate the invention.

Example in vivo animal experiment study of Effect of edaravone and Isochrysin on TBI rats

Preparation of experimental material and model

1. Material

Adult male clean grade SD rats weighing about 250 g. TUNEL apoptosis detection kit; TNF-alpha, IL-6, IL-1 alpha, IL-1 beta enzyme-linked immunosorbent assay (ELISA) kit; edaravone (edaravone injection: 5ml (10 mg)/zhi, tokyo Dongyuan pharmaceutical Co., Ltd.); isochrysol (isochrysol hydrochloride, sigma).

2. Laboratory animals and groups

Healthy male SD rats, weighing about 250g, 50, were randomly divided into five groups of 10 animals each, grouped as follows:

a first group: false operation group (Sham group)

Second group: TBI group (model control group)

Third group: edaravone group 3mg/kg

And a fourth group: isochrysin group 1mg/kg

And a fifth group: edaravone (3mg/kg) + isochrysol (1 mg/kg).

3. Preparation of animal experiment model

Animals were kept at 25 ℃ in 12h light and dark cycles with free diet and drinking water. Preparing a TBI model by adopting an improved Feeney DM free fall epidural impact method: after pentobarbital sodium abdominal anesthesia (50mg/kg), the scalp is cut at a position 2mm behind the right coronary suture and 2mm beside the midline, a bone hole with the diameter of 5mm is drilled, the dura mater is kept intact, a striking hammer with the weight of 30g freely falls from a position of 20cm to impact a striker to cause middle-sized craniocerebral injury of a rat (the injury impact force is 600g cm), bone wax seals the bone hole, and the scalp is sutured. Sham groups only open the bone window and do not hit. Starting 1h after injury, edaravone group was intraperitoneally injected with (IP) edaravone 3mg/kg (3mg/kg, IP), isochrysone group (1mg/kg, IP), combination group edaravone (3mg/kg, IP) + isochrysone (1mg/kg, IP), Sham group and model control group were Intraperitoneally (IP) injected daily with equal doses of saline. Each group was administered for 14 consecutive days.

Second, Experimental methods

1. Neurological impairment scoring

Rats were examined for motor, sensory and reflex using a modified neurological severity scale (mNSS). Scoring occurs when a task cannot be completed correctly or the attempted reflex disappears. Higher scores indicate more impaired neurological function. Neurological function assays were performed 1, 3, 7, 14 days after modeling, respectively.

2. Determination of water content in brain tissue

Rats 1, 3, 7 and 14 days after modeling are taken to take cerebral cortex (200 +/-20) mg at the position of about 2mm of the edge of a bone window, filter paper is used for sucking blood stains and cerebrospinal fluid on the surface of the brainstem, the rats are wrapped by known weight tinfoil, the rats are weighed by a wet weight on an electronic analytical balance, then the rats are placed in an oven at 100 ℃ for baking for 24 hours until the weight is constant, and then the dry weight is measured. Brain tissue water content (%) - (wet-dry weight)/wet weight 100%.

3. TUNEL staining for detection of neuronal apoptosis

Taking the cortex generated by cerebral contusion and laceration at the edge of a bone window as a specimen, carrying out enzymolysis on deoxyribonucleotide terminal transferase (TdT) according to the method steps shown by an apoptosis kit, carrying out labeling with digoxin marker, and developing color with DAB color developing agent. Positive for apoptosis: cell body shrinkage, irregular shape, cell nucleus shrinkage, and brown or brownish color. Counting adopts a double-blind method, apoptosis positive cells around brain injury are observed under an optical microscope high-power lens (400 times), 5 visual fields are randomly collected, the ratio of the apoptosis positive cells is counted, and an average value is taken for statistics.

4. Determination of inflammatory factor content in brain tissue

And detecting the content of the inflammatory factors by an ELISA method. Taking brain tissues of each group of mice modeling 3d after injury, homogenizing, adding samples into 96-well plates respectively coated with primary antibody, and measuring optical density (OD value) at the wavelength of 450nm according to the operation steps of a kit instruction. Drawing a standard curve, and calculating the contents of inflammatory factors TNF-alpha, IL-6, IL-1 alpha and IL-1 beta in the sample to be detected according to the standard curve.

Third, experimental results

1. Effect of composition of edaravone and isoxsuprine on nerve function of TBI rats

Evaluating the nerve functions of each group by using the mNSS, wherein the nerve function scores of the Sham group are not obviously changed before and after the operation, and the scores are 0-2 in 1-14 days; the damage of the model group is the most serious, and compared with the Sham group, the mNSS score of the rat is remarkably improved (compared with the Sham group, P is less than 0.001), thereby indicating that the modeling is successful. Compared with the model group, the edaravone group and the isochrysone group can reduce the mNSS score from day 3 (P <0.05 compared with the model group); the edaravone and isoxsuprine composition group was able to significantly reduce the mNSS score from day 3 (P <0.01 compared to the model group), and the results were also statistically different (P <0.05) compared to the edaravone group or the isoxsuprine group. The results are shown in Table 1.

TABLE 1 comparison of mNSS scores after TBI (x. + -. s) for groups of rats

P <0.05 for the Sham group, P <0.01 for the Sham group, P <0.001 for the Sham group; compared with the model group, P is less than 0.05, compared with the model group, P is less than 0.01; compared with the edaravone group, P of delta is less than 0.05, and compared with the edaravone group, P of delta is less than 0.01; p <0.05 compared to the isochrdrolin group, and P <0.01 compared to the isochrdrolin group.

The results show that the edaravone and the isoxsuprine both have a certain protection effect on the recovery of the nerve function of the rat after the TBI, the combination of the edaravone and the isoxsuprine can further promote the recovery of the nerve function of the rat after the TBI, and the combination of the edaravone and the isoxsuprine can play a synergistic effect to a certain extent.

2. Effect of composition of edaravone and isoxsuprine on brain tissue water content of TBI rats

Compared with the Sham group, the water content of brain tissue in the TBI group is obviously increased, the two groups have a remarkable statistical difference (P is less than 0.01), and the water content of the brain tissue is highest on the 3 rd day after injury and then gradually decreases. The effect of the isochrysq group on the water content of brain tissue was not statistically significant compared to the model group (P > 0.05); the edaravone group (P <0.05) and the composition group (P <0.01) showed a decrease in brain water content from day 3 compared to the model group and the isoxsuprine group, and had statistical differences. By day 14, the composition group had no statistical difference compared to the Sham group (P > 0.05). The results are shown in Table 2.

TABLE 2 comparison of TBI hindbrain tissue Water content for various groups of rats (x. + -. s,%)

P <0.05 for Sham group, P <0.01 for Sham group; compared with the model group, P is less than 0.05, compared with the model group, P is less than 0.01; compared with the edaravone group, P of delta is less than 0.05, and compared with the edaravone group, P of delta is less than 0.01; p <0.05 compared to the isochrdrolin group, and P <0.01 compared to the isochrdrolin group.

The results show that the edaravone can reduce the water content of the brain tissue of the rat after TBI, the isochrysone has little influence on the water content of the brain tissue of the rat after TBI, but can enhance the reduction effect of the edaravone on the water content of the brain tissue of the rat, and the edaravone and the isochrysone can obtain a synergistic effect on the reduction of the water content of the brain tissue of the rat after TBI when the edaravone and the isochrysone are combined.

3. Effect of edaravone and isoxsuprine composition on apoptosis of nerve cells of TBI rats

The TUNEL staining of brain tissue sections showed: the apoptosis of cerebral cortex in the TBI model group reaches a peak value 24h after injury, the apoptosis percentage is (25.37 +/-1.88)%, and apoptotic cells are still maintained at a higher level for 3-14 d. In sharp contrast to the apoptosis rate of Sham group (2.24 ± 0.61)% there was a very significant difference (P < 0.001). The change rule of the percentage of the apoptotic cells in the edaravone group along with time is similar to that in the TBI group, but the number of the apoptotic cells is obviously reduced (compared with the model group, P is less than 0.05); the isoxsuprine group itself had no statistical significance for the number of apoptotic cells (P >0.05 compared to the model group), but the edaravone and isoxsuprine composition group was able to further reduce the number of apoptotic cells (P <0.01 compared to the model group), and the reduction in the number of apoptotic cells in the composition group was also statistically different (P <0.05 or P <0.01) compared to the edaravone group.

Table 3 comparison of the percentage of apoptotic cells at each time point after TBI in each group of rats (x. + -. s,%)

The results show that although the isoxsuprine has no influence on the apoptosis of rat nerve cells after TBI, the isoxsuprine can enhance the anti-apoptosis effect of edaravone, and the composition of the isoxsuprine and the edaravone has obvious synergistic effect.

4. Effect of edaravone and isoxsuprine composition on inflammatory factor content in brain tissue of TBI rat

Compared with the Sham group, the brain tissues of rats in the model group are obviously increased in TNF-alpha, IL-6, IL-1 alpha and IL-1 beta (P is less than 0.01); compared with the model group, the contents of TNF-alpha, IL-6 and IL-1 alpha in the brain tissues of the rats in the edaravone group are reduced, and the statistical significance is achieved (P is less than 0.05); the isochryshulin group has a tendency of reducing inflammatory factors in brain tissues of rats, but has no statistical significance compared with a model group (P >0.05), the composition group can obviously reduce the contents of TNF-alpha, IL-6, IL-1 alpha and IL-1 beta in the brain tissues of rats, and the composition group has obvious difference compared with the model group (P <0.01) and the edaravone group (P < 0.05).

TABLE 4 comparison of inflammatory factor content (ng/L) (x. + -.s) in brain tissue at various time points after TBI in various groups of rats

The results show that after TBI, inflammatory cytokines in rat brain tissues such as TNF-alpha, IL-6, IL-1 alpha, IL-1 beta and the like are remarkably increased, edaravone can reduce the content of inflammatory factors, isocratin has a reduction trend on the content of inflammatory factors, but the statistical difference is not generated compared with a model group (P is greater than 0.05), but isocratin can remarkably enhance the inhibition effect of edaravone on the inflammatory factors, and the composition of the two has a remarkable synergistic inhibition effect on the generation of the inflammatory factors after TBI.

The result of the invention shows that although the isoxsuprine has no obvious influence on the apoptosis of the brain nerve cells of the rats after TBI and the expression of inflammatory factors in brain tissues, the isoxsuprine can enhance the anti-apoptosis and anti-inflammatory effects of the edaravone. The isochrysulvin is a cerebral vasodilator, and the effect is supposed to be related to the cerebral vasodilation of the isochrysulvin, so that the cerebral vasospasm after TBI is relieved, and the blood brain barrier permeation effect of edaravone is increased.

The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.

Claims

1. The application of the pharmaceutical composition in preparing the medicine for treating traumatic brain injury is characterized in that the pharmaceutical composition comprises edaravone, isochrysulfop and a pharmaceutically acceptable carrier, wherein the weight ratio of edaravone to isochrysulfop is 3:1, and the pharmaceutical composition is used for improving the nerve function after the traumatic brain injury, reducing the encephaledema degree after the traumatic brain injury, inhibiting the apoptosis of nerve cells and reducing the central inflammation reaction mediated by microglia.

2. The use according to claim 1, wherein the traumatic brain injury is selected from concussion, closed craniocerebral injury or open craniocerebral injury.

3. Use according to claim 1 or 2, characterized in that the pharmaceutical composition is in the form of a formulation selected from the group consisting of tablets, granules, capsules, suspensions, oral liquids, injections, transdermal patches, or inhalants.