WO2017190526A1 - Application of tetramethylpyrazine derivative tetramethylpyrazine nitrone in prevention and treatment of brain injury diseases - Google Patents

Abstract

The present invention belongs to the technical field of drugs, and relates in particular to an application of a tetramethylpyrazine derivative tetramethylpyrazine nitrone in the preparation of a drug and the prevention and treatment of brain injury diseases. Experiments show that tetramethylpyrazine nitrone can improve traumatic brain injuries and brain injuries after subarachnoid haemorrhages. Specifically, tetramethylpyrazine nitrone can improve the behaviour of model rats having traumatic brain injuries and subarachnoid haemorrhages, reduces the cerebral infarction area of a traumatic brain injury, improves the vasospasm state after a subarachnoid haemorrhage, and thus has a protective effect on brain tissue. The mechanism of action of tetramethylpyrazine nitrone is related to inhibiting oxidative stress injury, inhibiting inflammation and resisting apoptosis. Tetramethylpyrazine nitrone may be used as a drug which prevents or treats brain injury diseases, particularly traumatic brain injuries and subarachnoid haemorrhages, and may be made into a variety of dosage forms with a medicinal carrier.

Description

Application of Ligustrazine Derivative Nitrones in Prevention and Treatment of Brain Injury Diseases Technical field

The invention belongs to the technical field of medicine and relates to the application of the ligustrazine derivative nitroketazine in the prevention and treatment of brain injury diseases, in particular in the preparation of medicines and in the prevention and treatment of traumatic brain injury and subarachnoid hemorrhage diseases.

Background technique

Traumatic brain injury (TBI), also known as intracranial injury, is a common neurosurgical disease and is recognized as one of the most frequently-occurring and refractory injuries. A large number of studies have confirmed that brain dysfunction after TBI is not only due to the primary mechanical damage such as the initial mechanical damage, but also to the complex secondary neuron "second strike" that occurs after injury. Neuronal damage is related to calcium overload, excitatory amino acid toxicity, mitochondrial dysfunction, oxidative stress, apoptosis and inflammatory response.

As early as 1986, Kontos et al [J Neurosurg, 1986, 64 (5): 803-807] demonstrated the existence of a large amount of free radicals after brain injury. Later, studies have shown that oxidative stress plays an important role in the pathophysiological process after TBI. It is mainly characterized by an increase in free radical content in brain tissue, accumulation of peroxidation products, and reduction of antioxidant substances in the brain such as reduced glutathione, superoxide dismutase, and ascorbic acid. Oxidative stress can cause direct damage to nerve cells through lipid peroxidation, protein oxidation, nucleic acid oxidation, inflammation, and the like. Oxidative stress can also induce neuronal apoptosis indirectly through the mitochondria and nuclear transcription factor NF-κB. Therefore, inhibition of oxidative stress damage after brain trauma is an effective way to protect neurons.

Inflammation is an important component of the pathophysiology of traumatic brain injury. Due to the complexity of inflammatory response and the diversity of inflammatory factors, inflammation and cerebral edema formation, nerve cell death, and increased intracranial pressure are present in traumatic brain injury. close relationship. Inflammatory biochemical markers, which are directly derived from damaged brain cells, can be inferred from the changes in their indicators to predict the function and metabolism of brain tissue, so it is considered to be an important reference for clinical diagnosis and treatment. Inflammatory biomarkers produced by neurons or glial cells after traumatic brain injury include plasma S-100B, neuro-specific enolase (NSE), and myelin basic protein (myelin basic protein, MBP), glial fibrillary acidic protein (GFAP). GFAP is an intermediate filament protein that is abundantly present in the astrocyte cytoskeleton of the central nervous system. It has not been found in tissues other than the central nervous system, so GFAP has a high specificity. In the case of multiple wounds, its detection The role is particularly prominent. GFAP can not only determine the severity of the injury, but also assess the prognosis and mortality, and even predict the changes in intracranial pressure.

Secondary brain injury is mainly manifested in the apoptosis of nerve cells. If the apoptosis is excessive or does not interfere, the final nerve cell Death, impaired brain function. Under certain conditions, inhibition of excessive neuronal apoptosis after craniocerebral injury is a way of brain protection. It is one of the key topics in neurosurgical research to seek new neuroprotective drugs to prevent the occurrence of apoptosis, avoid secondary damage of nerve cells, and minimize brain damage.

Subarachnoid hemorrhage (SAH) refers to a brain injury caused by blood flow into the subarachnoid space after the blood vessels in the bottom of the brain or the surface of the brain are ruptured. The blood enters the subarachnoid space and is rapidly spread by cerebrospinal fluid surrounding the brain and spinal cord, stimulating the meninges to cause meningeal irritation. Increased amount of cranial content causes an increase in intracranial pressure and even cerebral palsy. Blood coagulated in the ventricles and brain base can block the cerebrospinal fluid circulation pathway, causing obstructive hydrocephalus caused by absorption and reflux, or causing arachnoid adhesions. The expansion or rupture of posterior communicating aneurysms can oppress adjacent oculomotor nerves. Produce varying degrees of oculomotor nerve palsy. The vasoactive substances released by blood cells can cause vasospasm, and severe cases of cerebral infarction occur. In the past few decades, vasospasm and aneurysm rupture and rebleeding have been considered a major factor in high mortality and high disability. However, multi-center clinical trials have shown that early brain injury after subarachnoid hemorrhage is the leading cause of death and disability. The molecular mechanisms of SAH brain injury include: calcium ion channel opening, free radical production, inflammatory reaction, apoptosis and necrosis. At present, the clinical treatment of SAH is very limited, only general treatment and symptomatic treatment, reduce intracranial pressure, prevent rebleeding. For early brain injury, there is still no effective treatment strategy.

Chuanxiong is a traditional Chinese medicine for promoting blood circulation and removing blood stasis. It has a thousand years of clinical application. It can promote blood circulation, relieve phlegm and relieve pain, and is used for stasis and blood stasis. Ligustrazine ( chemical name 2,3,5,6-tetramethylpyrazine, TMP) is the main active ingredient of Chuanxiong, and has rich pharmacological effects, including scavenging free radicals, blocking calcium channels, and anticoagulant thrombolysis. It can improve the microcirculation, relieve vasospasm, anti-inflammatory effect, inhibit apoptosis and neuroprotection, and is widely used in the treatment of cardiovascular and cerebrovascular diseases.

Zhang et al reported that TMP can directly scavenge free radicals and superoxide anions, increase the expression of antioxidant protein HO-1, enhance the activity of SOD and glutathione peroxidase, and inhibit the production of MDA in rats with ischemic brain injury. Thereby protecting cells from oxidative damage [Acta pharmacological Sinica, 1994, 15: 229-231]. Peter Kai et al [Planta Medica. 1996, 62-65] demonstrated that 10 μM of TMP can significantly inhibit L-type calcium channels and significantly inhibit KCl induction in primary cultured rat hippocampal neurons. Intracellular calcium is elevated in the presence of extracellular calcium and calcium free fluid. In the rat model of ischemic injury, TMP can inhibit neuronal apoptosis, which is related to the inhibition of the expression of the pro-apoptotic protein Bax and the up-regulation of the expression of the anti-apoptotic protein Bcl-2. In addition, in vitro cell experiments have shown that TMP can inhibit H ₂ O ₂ -induced apoptosis, and its mechanism is also related to inhibition of Caspase-3 activation and cytochrome C release [BMC Neurosci, 2006, 7:48].

Studies by Liao and Kao et al have demonstrated that TMP can reduce cerebral infarction size and cerebral edema in rats with focal cerebral ischemia and improve animal behavior, and its mechanism of action is related to inhibition of cellular inflammatory response [Neurosci Lett, 2004, 372(1-2): 40-45; Exp Neurol, 2013, 247: 188-201]. Lu et al. showed that TMP has a significant protective effect on MPTP-induced Parkinson's disease animal model, and has obvious protective effect on dopamine neurons, which promotes the expression of anti-apoptotic protein Bcl-2 and inhibits pro-apoptotic protein. Bax expression is associated with inhibition of cytochrome C release [Int J Biol Sci, 2014, 10(4): 350-357].

Zhu et al studied the effect of TMP on blood rheology in patients with acute craniocerebral injury. The blood viscosity of patients with acute craniocerebral injury was significantly increased. The patient was given TMP injection 80 mg/d. After 7 days of continuous administration, the patient's red blood cell deformability was significant. Increased, blood viscosity decreased, brain tissue ischemia and hypoxia and cerebral edema were alleviated. Chen Ying studied the efficacy and time window of TMP in traumatic brain injury. Patients in the treatment group received TMP 5% glucose injection, 80 mg/day, for 5 days after 1-4h or 4-8h. After 5 days, the patient's venous blood was collected, and plasma NO and SOD levels were determined by radioimmunoassay. The study found that after TMP treatment, the patient's NO level was significantly reduced, while the level of SOD was significantly increased, and the efficacy of administration at 1-4 h after onset was better than that of 4-8 h. The protective effect of TMP on traumatic brain injury is related to the inhibition of free radical damage.

Shao et al studied the effect of TMP on cerebral vasospasm after subarachnoid hemorrhage in rabbits. The results showed that TMP significantly reduced the cerebral vasospasm after subarachnoid hemorrhage by stimulating eNOS expression and promoting NO production [Brain Res] .2010, 1361: 67-75]. Gao et al. administered intravenous injection of 30 mg/kg TMP to SAH model rats. After 24 hours, the effects of TMP on brain edema, behavior and cerebral vasospasm were observed. The results demonstrate that TMP significantly improves the behavior of SAH model rats, reduces brain edema, and improves sputum vasospasm. Its protective effect may be related to inhibition of Caspase-3 mediated apoptosis [Auton Neurosci, 2008, 141(1- 2): 22-30].

Nitronones are a class of compounds with strong free radical scavenging ability. It has been found that nitrone compounds have certain therapeutic effects on cancer, stroke, and Parkinson's disease. A typical representative of such drugs is NXY-059. A large number of preclinical animal experiments, as well as phase II clinical trials and the first phase III clinical trials have demonstrated that NXY-059 has a good therapeutic effect on ischemic stroke. However, in the second phase III clinical trial, NXY-059 did not. Achieving the desired therapeutic effect, and ultimately failing, one of the reasons for its failure may be related to its difficulty in passing the blood-brain barrier.

Summary of the invention

It is an object of the present invention to provide a novel use of a ligustrazine derivative, nitroketazine, in the preparation of a medicament and in the prevention and treatment of a brain injury disease.

In order to overcome the defect that NXY-059 is difficult to pass the blood-brain barrier, the present invention replaces the sodium benzenedisulfonate structure in the NXY-059 structure with a TMP structure, and obtains a TMP nitrone compound nitroketone which easily passes through the blood-brain barrier. Research of the invention It was found that nitroketazine has a good protective effect on animal models of TBI and SAH brain injury, and its mechanism is related to inhibition of oxidative stress injury, inhibition of inflammatory reaction and apoptosis.

The ligustrazine derivative of the present invention is a coupling of ligustrazine and nitrone, and its structural formula is as follows:

The ligustrazine derivative nitroketone of the present invention can be used for the preparation of a medicament which can be used for the prevention and treatment of diseases of brain damage. The brain injury diseases include, but are not limited to, traumatic brain injury and subarachnoid hemorrhage. For example, the traumatic brain injury is a closed head injury or an open brain injury.

In the preparation of a medicament for the prevention and treatment of a brain injury disease, the nitroketazine may be used alone or in combination with other drugs.

The nitroketazine can be mixed or dissolved with any usable carrier, such as a pharmaceutically, mucosal, parenteral or parenterally administrable pharmaceutical carrier, which is used in the form of a conventional preparation such as tablets or granules. , injections, powders, capsules, suspensions. Pharmaceutically acceptable excipients and additives for use in the medicament include non-toxic compatible fillers, binders, disintegrants, buffers, preservatives, antioxidants, lubricants, flavoring agents, thickening Agent, colorant, emulsifier or stabilizer. The drug can be prepared in a conventional process for various formulations.

According to various embodiments, the present invention also provides an amount of the nitroketopazine for preventing and treating brain damage diseases. The amount includes 0.01-100 mg/kg body weight, or 1-100 mg/kg body weight or 10-100 mg/kg body weight.

According to a specific embodiment of the present invention, the inventors conducted a rigorous demonstration of the efficacy of the ligustrazine derivative nitroketone by animal experiments. The effects and benefits of the present invention include the following:

(1) The present invention finds a new use of the ligustrazine derivative nitroketone, which is a preparation of a medicament and is used for preventing and treating brain injury diseases;

(2) The ligustrazine derivative of the present invention has a significant advantage in the treatment of brain injury diseases, and has multiple effects such as free radical scavenging, inhibition of inflammation and anti-apoptosis, and inhibition of neurons. The multiple factors of injury are better than the single drug. On the other hand, compared with NXY-059, nitroketone has a greatly improved ability to pass the blood-brain barrier, which is very beneficial for the treatment of brain injury diseases.

(3) The ligustrazine derivative of the present invention has a significant neuroprotective effect, and it is possible to prevent the occurrence of brain injury diseases and delay and prevent the development of diseases. In contrast, existing drugs for treating brain damage diseases, such as nimodipine, which treats subarachnoid hemorrhage, can only improve symptoms and prevent the occurrence and development of diseases.

DRAWINGS

The invention will now be further described with reference to the accompanying drawings and embodiments.

Figure 1: Effect of nitroketazine (TBN) on behavior in TBI rats. (A) Effect of nitroketazine on rod capacity in TBI rats; (B) Effect of nitroketazine on TBI rats in balance beam test; (C) Effect of nitroketazine on TBI rats in NSS test; (D) The effect of nitroketazine on the ability of TBI rats to contact the adhesive strip in the adhesive strip test; (E) the effect of nitroketazine on the ability of TBI rats to tear off the adhesive strip in the adhesive strip test. P < 0.05, ^### P < 0.01 and ^### P < 0.001 compared with the control group (Sham); ^* P < 0.05, ^** P < 0.01 and ^*** P < 0.001 compared with the model group.

Figure 2: Results of the effects of nitroketazine (TBN) on histological changes in cerebral infarction in TBI rats. Nitronone inhibits the activation of microglia and prevents the formation of glial scars in glial cells, thereby reducing the inflammatory response.

Figure 3: Results of the effects of nitroketazine (TBN) on the expression of neuronal (NeuN) and glial fibrillary acidic protein (GFAP) in TBI rats. ^### P<0.001 was compared with the control group (Sham); ^* P<0.05 and ^*** P<0.001 compared with the model group.

Figure 4: Results of the effects of nitroketazine (TBN) on the expression changes of 4-hydroxynonenal (4-HNE) and 8-hydroxydeoxyguanosine (8-OHdG) in TBI rats. ^### P<0.001 was compared with the control group (Sham); ^* P<0.05 and ^** P<0.01 compared with the model group.

Figure 5: Effect of nitroketazine (TBN) on the expression of Bcl-2, Bax and Caspase-3 in Caspase apoptosis pathway in TBI rats. ^# P <0.05 and ^### P <0.001 with the control group (Sham) comparison; ^* P <0.05 compared and ^*** P <0.001 with model group.

Figure 6: Effect of nitroketazine (TBN) on the expression of Nrf-2 and HO-1 protein in Nrf-2/ARE signaling pathway in TBI rats. ^# P<0.05, ^### P<0.01 and ^### P<0.001 were compared with the control group (Sham); ^* P<0.05 and ^** P<0.01 compared with the model group.

Figure 7: Effect of nitroketazine (TBN) on behavioral impairment in SAH rats.

Figure 8: Effect of nitroketazine (TBN) on the severity of bleeding in SAH rats. ^* P < 0.05 and ^** P < 0.01 compared to the model group.

Figure 9: Effect of nitroketazine (TBN) on vasospasm in SAH rats. ^* P < 0.05 and ^** P < 0.01 compared to the model group.

detailed description

The invention is explained in detail in the examples given below. However, the examples are merely illustrative and are not to be construed as limiting the scope of the invention.

Example 1. Effect of ketidazine on behavior of TBI rats

Rat TBI model was established by PCI3000 instrument. 90mg/kg ketidazine was injected into the tail vein at 3h, 6h and day2-day7 after operation. The behavioral scores were performed at 3h, 24h, 72h, 5d and 8d. (rotating bar test, strip adhesion test, NSS behavioral score, balance beam test).

Rotating rod test: The rod tester was used for the test. The SD rats were recorded daily to accelerate the roller from 6 rpm to 30 rpm (every 5 rpm, interval 10 s, until 30 rpm, stay until the end The process is 120s) and the axis remains unsuccessful. The next test is performed every 15 minutes for a total of 2 times. Animals were trained for 3 days prior to modeling and rats in the bar for more than 75 s were included in the experimental group.

Paper strip adhesion test: a sleeve is made of a paper strip, which is wound around the forelimb of the animal, and is connected back and forth to form a circle, and the finger end can be exposed a little from the sleeve. The following indicators are timed separately. The first item starts from the time the animal is returned to the cage. The second item starts timing only when the animal begins to try to tear off the sleeve until the sleeve is torn off. The ipsilateral and contralateral limbs of the lesion were tested independently and repeated twice a day. Animals were trained for 3 days in advance of modeling, and animals that could tear off the paper within 1 minute were included in the experimental group.

Balance beam test: The instrument used in the balance beam walking test consists of a square crossbar (2.5 cm wide) and a black box. The crossbar is connected to the black box. The whole experiment is carried out in a dark environment. Only a strong light is placed above the starting point to promote the rat. Climb over the crossbar and repeat 3 times, with the average as the final result. Animals were trained for 3 days in advance of modeling, and animals that passed the balance beam were included in the experimental group. The specific scores are shown in Table 1.

Table 1. Score sheet for assessing balance ability

Nerve damage index: The behavioral deficit of the rat was assessed using the Neurological Severity Score scale, as shown in Table 2.

Table 2. Neurological damage severity score

Example 2. Effect of nitroketazine on histological changes in cerebral infarction in TBI rats

On the 8th day after operation, perfusion internal fixation was performed, and the brain tissue was taken out and further fixed, dehydrated and paraffin-embedded, and sliced and subjected to paraffin-HE staining. The process is as follows: 1) Dewaxation of xylene is transparent: 5 times each time, 2 times each; 2) Alcohol gradient elution: 100% alcohol (5 min); 100% alcohol (2 min); 95% alcohol (2 min); 95% alcohol (2min); 3) washed distilled water: deionized water, 1min; 4) hematoxylin staining for 15min, 37 ° C; 5) washing: washed glass slides 30s; 6) differentiation: 1% hydrochloric acid alcohol differentiation 3s; 7) washing : Circulating water for 10-20min (12min), this step is blue; 8) Eosin staining: 0.5% eosin staining for 15min, 37°C; 9) Alcohol hydration: 70%, 80%, 95%, 100 in order %, 100%, 100% alcohol in hydration for 30s, 30s, 30s, 1min, 3min, 3min; 10) Transparency: xylene transparent 2 times, each time 3min; 11) Cover: neutral resin sealing, taking pictures Observed.

Example 3. Effect of nitroketazine on the expression of neurons, glial fibrillary acidic protein, 4-hydroxynonenal and 8-hydroxydeoxyguanosine in TBI rats

On the 8th day after operation, perfusion internal fixation was performed, and the brain tissue was taken out and further fixed, dehydrated and paraffin-embedded, and sliced and subjected to paraffin-immunohistochemical staining. The procedure is as follows: 1) Dewaxation of xylene is transparent: 10 min each time, 2 times; 2) Gradient elution: 100% ethanol (5 min); 100% ethanol (2 min); 95% ethanol (2 min); 95% ethanol (2 min) ); deionized water rinse; 3) section antigen repair: section repair using citrate buffer (pH = 6.0) microwave method for antigen retrieval (high fire 5min, medium and low fire 10min), naturally cooled to room temperature; 4) TBS wash Tablet: Wash with 1*TBS, 3 min each time, 3 times; 5) 3% H ₂ O ₂ (TBS configuration) closed: 10 min in the dark, rinse with deionized water; 6) TBS wash: 3 min each time 3 times; 7) Closure: dry the water around the sliced tissue with filter paper, circle with immunohistochemical pen, then add 10% FBS (TBS configuration) and incubate for 2 hours at room temperature; 8) Wash: 3 min each time 3 times; 9) Adding antibody: Add primary antibody: Neu (1:500), GFAP (1:400), 4-HNE (1:200), 8-OHdG (1:200), incubate overnight at 4 °C . The primary antibody was removed the next day, and the sections were gently placed in the TBS solution for 3 times for 3 minutes each time. Then, add horseradish peroxidase-labeled secondary antibody (provided in DAB kit), incubate for 1 h at room temperature; 10) Color development: TBS wash 3 times, 3 min each time, add DAB color provided by the kit Working solution, incubate for 5 min at room temperature, microscopically control dyeing depth; 11) Gradient alcohol hydration: alcohol concentration from 95%, 95%, 100%, 100% gradual hydration, 2 min each time; 12) Cover: xylene transparent, Neutral gum seals and then photographed.

Example 4. Effect of nitroketazine on the expression of Caspase apoptosis pathway (Bcl-2, Bax, Caspase-3) and Nrf-2/ARE signaling pathway (Nrf-2, HO-1) in TBI rats

After the brain tissue was weighed, tissue lysate was added in a volume ratio of 1:10 to the lysate according to the weight of the tissue, and placed on an ice bath to homogenize with a tissue homogenizer. The tissue homogenate was collected in a 1.5 mL EP tube and centrifuged at 12000 rpm for 15 min at 4 ° C using a high-speed centrifuge. After centrifugation, the supernatant was carefully taken as the cortical tissue protein. The supernatant was dispensed and stored frozen at -80 °C.

Western blotting was carried out according to a conventional experimental method in which the primary antibody was diluted 1:1000 and the secondary antibody was diluted 1:2000.

Example 5. Effect of nitroketazine on behavior of SAH rats

The SAH model was induced by suture method. 60mg/kg ketidazine was injected twice a day at 3h, 6h and day2-day7, and neurobehavioral evaluation was performed at 3h, 24h, 72h, 5d and 8d. . The evaluation method is shown in Table 3.

Table 3. Neurological damage severity score

Example 6. Effect of nitroketazine on bleeding in SAH rats

At the end of the experiment, the rats were euthanized with pentobarbital, the entire brain tissue (including the cerebellum and brainstem) was taken out, photographed, and then the picture was divided into 6 parts. Each part was subjected to the SAH severity score according to the following method. Total score: 18 points: 0 points: no subarachnoid hemorrhage; 1 point: a small amount of bleeding; 2 points: moderate bleeding, but the blood vessels can still be clearly identifiable; 3 points: severe bleeding, blood vessels can not be distinguished. Among them, 0-7 is divided into mild hemorrhage; 8-12 is divided into moderate hemorrhage; 13-18 is divided into severe hemorrhage.

Example 7. Improvement of vasospasm on vasospasm in SAH rats

After 7 days of model administration, rats were euthanized by pentobarbital sodium anesthesia, brain tissue photographed, fixed in 4% paraformaldehyde, pathologically sectioned after paraffin embedding, HE staining of basilar artery, analysis of basilar artery circumference Length and thickness.

Claims (8)

1. Use of a ligustrazine derivative, nitroketone, for the preparation and use of a medicament for the prevention and treatment of a brain injury disease, the structural formula of the nitroketazine being as follows.

   
2. The use according to claim 1, wherein the brain injury disease is traumatic brain injury or subarachnoid hemorrhage.
3. The use according to claim 2, wherein the brain injury disease is a traumatic brain injury.
4. The use according to claim 3, wherein the traumatic brain injury is a closed head injury or an open head injury.
5. The use according to claim 2, wherein the brain injury disease is subarachnoid hemorrhage.
6. The use according to any one of claims 1 to 5, wherein the nitroketazine is used alone or in combination with other drugs.
7. The use according to any one of claims 1 to 5, wherein the nitroketazine and a pharmaceutically acceptable carrier are in various dosage forms, which are tablets, granules, injections, powders, capsules or suspensions.
8. The use according to claims 1-5, wherein the medicament is used in an amount of from 0.01 to 100 mg/kg body weight, or from 1 to 100 mg/kg body weight or from 10 to 100 mg/kg body weight.

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