阅读说明：本技术 苯并二呋喃酮类化合物在治疗难治性癫痫中的应用 (Application of benzodifuranone compound in treating intractable epilepsy ) 是由 邢春叶 孙鹏 范欣 于 2021-10-12 设计创作，主要内容包括：本发明公开了一种苯并二呋喃酮类化合物在制备治疗难治性癫痫药物中的应用,属于医药技术领域。本发明首次提出将苯并二呋喃酮类化合物用于治疗癫痫,尤其为难治性癫痫的治疗提供了新的候选药物。另外,本发明还提供了苯并二呋喃酮类化合物与丙戊酸钠联用的复方药物,二者在治疗难治性癫痫疾病中具有明显的协同效果。(The invention discloses an application of a benzodifuranone compound in preparation of a drug for treating intractable epilepsy, and belongs to the technical field of medicines. The invention firstly provides the application of the benzodifuranone compound in treating epilepsy, and particularly provides a new candidate drug for treating intractable epilepsy. In addition, the invention also provides a compound medicine combining the benzodifuranone compound and sodium valproate, and the compound medicine and the sodium valproate have obvious synergistic effect in treating intractable epilepsy.)

1. The application of the benzodifuranone compound in preparing the medicine for treating the epilepsy of the mammal, wherein the chemical structural formula of the benzodifuranone compound is as follows:

2. use of benzodifuranones according to claim 1, in the manufacture of a medicament for the treatment of epilepsy in a mammal, wherein the epilepsy is refractory epilepsy.

3. Use of the benzodifuranones according to claim 1 in the manufacture of a medicament for the treatment of epilepsy in a mammal, wherein the mammal is selected from rat, monkey, dog or human.

4. The pharmaceutical composition for treating intractable epilepsy is characterized in that active ingredients in the pharmaceutical composition consist of a benzodifuranone compound and sodium valproate, wherein the chemical structural formula of the benzodifuranone compound is as follows:

5. the pharmaceutical composition for treating refractory epilepsy according to claim 4, wherein the dosage ratio of the benzodifuranone compound to the sodium valproate is 1: 10 to 40.

6. The pharmaceutical composition for treating refractory epilepsy according to claim 5, wherein the dosage ratio of the benzodifuranone compound to the sodium valproate is 1: 20.

7. the pharmaceutical composition of claim 4, wherein the pharmaceutical composition is a formulation for gastrointestinal administration.

8. The pharmaceutical composition of claim 7, wherein each unit dosage form comprises 3.2-12.5 mg of the benzodifuranones and 125mg of sodium valproate.

9. The pharmaceutical composition of claim 8, wherein each unit dosage form comprises 5-8 mg of the benzodifuranones and 125mg of sodium valproate.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an application of a benzodifuranone compound in preparation of a medicine for treating intractable epilepsy.

Background

Epilepsy is a common nervous system disease, is a chronic recurrent transient brain dysfunction syndrome, and is characterized by epileptic seizures caused by abnormal discharge of cerebral neurons. Most patients are well controlled with Anti-Epileptic Drugs (Anti-Epileptic Drugs), but more than 30% of patients are ineffective with existing Anti-Epileptic Drugs, known as Refractory Epilepsy (RE). The international anti-epileptic union defines RE as the choice of more than or equal to 2 kinds of tolerable anti-epileptic drugs which are treated by sufficient dose and course of treatment and can not control the epileptic seizure after reaching effective concentration in vivo. While the domestic diagnosis of RE means that no central nervous system space-occupying lesion or other central nervous system diseases, frequent epileptic seizures, seizures are more than or equal to 4 times per month, observation is carried out for 2 years, proper first-line antiepileptic drugs are applied for formal treatment, the blood concentration of the drugs is in an effective range, the seizures cannot be controlled, and daily life is influenced.

The pathogenesis of epilepsy is very complex, and all the mechanisms of epilepsy are still not completely understood, but more and more learners consider epilepsy to be a neural network disease characterized by recurrent seizures. The neural network hypothesis suggests that signal molecules in the neural network can induce abnormal growth of axons of epileptics, including axon sprouting, synaptic reorganization, neurogenesis, gliosis and the like, and the above changes can promote the formation of abnormal neural networks, which not only inhibit endogenous anti-epileptic systems, but also prevent traditional anti-epileptic drugs from entering neuronal excitation targets, and finally cause RE to occur [ Tang, F, Hartz, AMS, Bauer, B.drug-residual epiilepsy: multiple hypotheses, few answers.Front neuron 2017; 8:301.]. In addition, frequent seizures can cause progressive brain plasticity changes, forming abnormal neural networks, leading to refractory epileptogenesis. Jiang et al [ Jiang LW, Qian RB, Fu XM, Zhang D, Peng N, Niu CS, Wang YH. alternative authentication networks and DMN in regenerative simplicity: A restriction-state functional and practical connectivity study. Epilepsy brand 2018 Nov; 88:81-86PMID:30243110] functional MRI data analysis of RE patients and healthy people in a resting state shows that the neural network connection function of the RE patients is reduced, and epileptic seizures can interfere with the interaction of the brain neural network and further influence information exchange of neurons. Although neural network hypothesis cannot explain the pathogenesis of all REs, a new direction is opened for the in-depth exploration of RE pathogenesis. The future deep research on the function of the abnormal neural network in the RE has important significance for attacking the RE and improving the life quality of RE patients.

At present, the treatment of epilepsy is mainly based on medicines, but for intractable epilepsy patients, the existing antiepileptic medicines are ineffective, and although proper surgical treatment is also used for relieving the seizures of the patients, the problems of surgical indications, high risk, high cost and the like cause the epilepsy to be greatly limited. Therefore, there is a great need to develop targeted antiepileptic drugs for different mechanisms of action. CN111499649A reports that an isolated benzodifuranone compound Asperterreurone A was extracted from Aspergillus terreus CC-S06-18 metabolite, and found to have cytotoxic effect on gastric cancer cell lines and induce cell cycle arrest and apoptosis.

Disclosure of Invention

The inventor summarizes and discovers in clinical work for years that reactive gliosis is one of the pathomorphological characteristics of epilepsy, and the inventor further discovers that the benzodifuranone compound Aspeterreurone A can relieve the spinal cord ischemia-reperfusion injury of rats by inhibiting neutrophil infiltration, micro-colloid cell activation and reactive gliosis. Therefore, the inventor of the invention conducted preliminary research on Asperterreurone A as a potential drug for treating intractable epilepsy, and found that the compound has an obvious anti-epilepsy effect on a lithium chloride-pirocarpine hydrochloride intractable epilepsy model rat, and is expected to be developed into a drug for treating intractable epilepsy.

In order to further expand more application fields of the benzodifuranone compound, the invention aims to provide application of Asperterreurone A of the benzodifuranone compound in preparing a medicament for treating epilepsy (refractory epilepsy) of mammals, wherein the chemical structural formula of Asperterreurone A is as follows:

further preferably, the mammal is selected from rat, monkey, dog or human.

In addition, the invention also provides a pharmaceutical composition for treating refractory epilepsy, and the active ingredients in the pharmaceutical composition consist of the benzodifuranone compounds and sodium valproate, so that the refractory epilepsy is treated synergistically.

Further preferably, the pharmaceutical composition for treating refractory epilepsy as described above, wherein the dosage ratio of the benzodifuranone compound to the sodium valproate is 1: 10 to 40. Still further preferably, the dosage ratio of the benzodifuranone compound to the sodium valproate is 1: 20.

further preferably, the pharmaceutical composition for treating refractory epilepsy is prepared into a preparation for gastrointestinal administration by adding pharmaceutically acceptable auxiliary materials.

Further preferably, each unit preparation of the pharmaceutical composition for treating intractable epilepsy comprises 3.2-12.5 mg of the benzodifuranone compound and 125mg of sodium valproate. Still more preferably, each unit preparation contains 5-8 mg of the benzodifuranone compound and 125mg of sodium valproate.

Compared with the prior art, the invention firstly provides that the benzodifuranone compound Aspeterreurone A is used for treating epilepsy, and particularly provides a new candidate medicine for treating intractable epilepsy. In addition, the invention also provides a compound medicine of Aspetereurone A and sodium valproate, and the Aspetereurone A and the sodium valproate have obvious synergistic effect in treating intractable epilepsy.

Detailed Description

The advantages and features of the present invention will become more apparent to those skilled in the art from the following description, taken in conjunction with the accompanying drawings, illustrating by way of example the principles of the invention. It should be understood that the illustrated embodiments are exemplary only, and are not intended to limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit of the invention, and it is intended to cover all such changes and modifications as fall within the scope of the invention. In addition, in the following examples, the acronym AtrA is used instead of AspeterreuroneA, the compound benzodifuranone, VPA, is used instead of sodium valproate.

Example 1 Effect of Asperterreuronea on refractory epilepsy cell model

Hippocampal neurons cultured up to day 7 were randomly grouped into a normal control group (group C), a model group (group M), a model +0.1 μ MAtrA group (group AtrA 1), a model +1 μ MAtrA group (group AtrA 2), a model +10 μ MAtrA group (group AtrA 3), and a model +100 μ MAtrA group (group AtrA 4). And culturing the normal control group change solution for 3 hours in a normal extracellular fluid, then recovering to 2mL of maintenance medium for culture, culturing the model group change solution for 3 hours in a magnesium-free extracellular fluid, then recovering to 2mL of maintenance medium for culture, culturing the AtrA group change solution with each concentration in a magnesium-free extracellular fluid for 3 hours, then recovering to the maintenance medium containing the final concentrations of 0.1 mu M, 1 mu M, 10 mu M and 100 mu MAspeterreone A, and putting the six-hole plate back to the culture box for continuous culture for 24 hours.

Maintaining the composition and preparation of a culture medium: neuron-specific neurobasal medium 98% (V/V) + B27 supplement 2% (V/V) +0.2 mol/L-glutamine. Sealing the bottle mouth with sealing film, and storing in refrigerator at 4 deg.C for use.

The magnesium-free extracellular fluid comprises the following components in parts by weight: A1L measuring cup is put into a rotor, 800mL of double distilled water is put into the measuring cup and placed on a magnetic stirrer, and the following medicines are accurately weighed by an electronic balance: 145mM NaCl, 2.5mM KCl, 10mM HEPES, 2mM CaCl₂10mM glucose and 0.002mM glycine are fully stirred until dissolved, the pH value is adjusted to 7.2 by NaOH, the volume is fixed to 1000mL by double distilled water, a filter is added with a 0.22 mu m filter membrane and wrapped by tinfoil paper, a sealing film of a cleaned glass bottle is sealed, the filter and the glass bottle are sterilized at high temperature and high pressure, the sealing film seals the bottle mouth, and the glass bottle is placed in a refrigerator at 4 ℃ for storage for standby. Normal extracellular fluid was magnesium-free extracellular fluid supplemented with 1mM MgCl.

The hippocampal neurons which are just separated and planted are round, transparent and have no protrusions, and are in a single cell suspension state. On the 3 rd day of culture, adherent hippocampal neurons extend to form several protrusions with uneven thickness, the cell bodies are transparent and are in fusiform, triangle and the like, and adjacent neurons can be connected by sparse networks. The hippocampal neurons cultured to the 5 th day have good growth state, obvious stereoscopic impression and refractivity, enlarged cell bodies and prolonged protrusion and interweaved into nets. Culturing hippocampal neurons at day 7, with enlarged and plump cell bodies and obvious halo; the number of neuron processes is increased, the axon is slender, and the processes among the neurons are mutually interwoven to form a dense, complex and uniformly distributed neural network.

After 24h of modeling (i.e., day 8 of culture), the normal control group (C) neurons were full and interwoven into a complex but evenly distributed and ordered neural network. After 24h of modeling, the hippocampal neurons of the model group (M) migrated, fused, dead neurons floated, and the neural network became cluttered and unevenly distributed. After 3h of modeling is carried out, the solution is changed into a normal maintenance culture medium, 0.1 mu M, 1 mu M, 10 mu M and 100 mu M of AtrA are added, the neuron state is improved to different degrees after 24h of culture, the fused neurons are reduced to different degrees, the neural grid distribution is more uniform, and obvious effects can be seen in 10 mu MAtrA groups and 100 mu MAtrA groups (AtrA3 group and AtrA4 group). Adding MTT20 μ L with final concentration of 0.5g/L, terminating the culture after 4h, removing culture solution in the well by suction, adding 150 μ L dimethyl sulfoxide into each well, and placing on a shaker for 10min under low speed oscillation. The absorbance (A) of each well was measured at 490nm in an ELISA and the relative viability of cells protected with Aspeterreurone A was calculated after A was determined according to the following formula: relative cell viability (%) ═ AtrA group a value-M group a value)/(C group a value-M group a value).

Since the a value of each well is proportional to the viability of the cells, the relative viability of the cells of the AtrA1, AtrA2, AtrA3, and AtrA4 groups was 4.1%, 62.9%, 94.4%, and 95.0%, respectively, as determined by a value measurement and calculation. This shows that 1-100. mu.M Aspeterreurone A has obvious protective effect on neuronal cells, especially 10-100. mu.M Aspeterreurone A can significantly inhibit neuronal damage in refractory epileptic cell models.

Example 2: study of efficacy of Asperterreurone A on Li-pilocarpine refractory epilepsy rats

Pilocarpine is a muscarinic receptor agonist that activates cholinergic neurons and disrupts the balance of glutamate and gaba. While lithium chloride enhances the function of pilocarpine. Temporal lobe epilepsy is the most common refractory epilepsy, and the lithium-pilocarpine model is often used to study temporal lobe epilepsy. The pathological features of the lithium-pilocarpine model are the production of inflammation, the proliferation of glial cells, the massive loss of neurons and the sprouting of moss fibres, which are similar to the neuropathological features of temporal lobe epilepsy. Therefore, the invention selects the lithium-pilocarpine rat model to carry out pharmacodynamic experimental study.

Healthy male SD rats, body mass in the range of 180-220 g. After the experiment begins, firstly, a lithium chloride-pirocarpine hydrochloride refractory epilepsy model is established, and the method comprises the following steps: 120mg/kg of lithium chloride is injected into the abdominal cavity, 1mg/kg of atropine is injected after 20 hours, and 50mg/kg of pilocarpine is injected into the abdominal cavity after 30 min. If the status of epilepsy does not appear, the intraperitoneal injection of the pilocarpine 10mg/kg is continued every 30min until the rats have 4-5-grade epilepsy and the status of epilepsy lasts for 60min, and the intraperitoneal injection of the chloral hydrate 300mg/kg stops the seizure.

The acute stage of epileptic seizure is maintained for 24h, then the incubation period of about two weeks is started, and 5% glucose and sodium chloride injection is given to the abdominal cavity for injection every day, 2 times/d and 5 ml/time, so as to supplement nutrition. Two weeks later, the rats successfully molded (implementing the Racine standard, with grade 4 or more being a successful epilepsy model) were randomly assigned to the model control group (M), AspeterreuroneA group (AtrA), sodium valproate group (VPA), and combination group (AtrA-VPA), with 8 rats each. Another 8 unmolded normal rats were used as a blank control group (C). Gavage was then initiated, and the specific doses given are shown in table 1, for a total of 6 weeks. The total number of seizures and total time of seizures in rats within 6 weeks after treatment were recorded since the start of drug treatment, and the statistical results for each group are shown in table 2.

Table 1: grouping and administration of laboratory animals

Table 2: comparison of Total number of epileptic seizures and Total time in rats of each group

In comparison with the set of M,^*p is less than 0.05; in comparison with the group of AtrA,^#p is less than 0.05; in comparison with the VPA group,^￥P＜0.05。

the experiment shows that the blank control group of rats has no phenomenon of epileptic seizure, the total number of epileptic seizures of the model-making rats is obviously increased, and the duration of the epileptic seizure is obviously prolonged. As can be seen from the statistical results of the tests in Table 2, compared with the model control group, each administration group can reduce the total times of the epileptic seizures of the rats to a certain extent and simultaneously shorten the total time of the epileptic seizures. Particularly, compared with the AtrA group and the VPA group, the total times of the epileptic seizures of the rats after the combined administration of the AtrA-VPA group are lower (P is less than 0.05), the duration of the epileptic seizures is shorter (P is less than 0.05), and the difference is statistically significant. The specific analysis of the experimental results can show that the anti-epileptic effect of Asperterreurone A combined with sodium valproate is obviously enhanced, the anti-epileptic curative effect is greater than the sum of the two, and the Asperterreurone A combined with sodium valproate has a remarkable synergistic effect.