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WO2024260261A1 - Utilisation de voie de signalisation bdnf-trkb dans la prévention et/ou le traitement de l'épilepsie liée à un trouble de déficience en cdkl5 - Google Patents

Utilisation de voie de signalisation bdnf-trkb dans la prévention et/ou le traitement de l'épilepsie liée à un trouble de déficience en cdkl5 Download PDF

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WO2024260261A1
WO2024260261A1 PCT/CN2024/097792 CN2024097792W WO2024260261A1 WO 2024260261 A1 WO2024260261 A1 WO 2024260261A1 CN 2024097792 W CN2024097792 W CN 2024097792W WO 2024260261 A1 WO2024260261 A1 WO 2024260261A1
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cdkl5
mice
epilepsy
bdnf
signaling pathway
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Chinese (zh)
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熊志奇
朱姊艾
李奕彦
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Center for Excellence in Brain Science and Intelligence Technology Chinese Academy of Sciences
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Center for Excellence in Brain Science and Intelligence Technology Chinese Academy of Sciences
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/553—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants

Definitions

  • the present invention relates to the field of biopharmaceuticals, and in particular, to the application of BDNF-TrkB signaling pathway in preventing and/or treating epilepsy associated with CDKL5 deficiency.
  • CDKL5 deficiency disorder is a rare genetic disease characterized by seizures that begin in infancy and are accompanied by significant developmental delays in multiple systems. CDKL5 deficiency occurs in approximately 1 in 40,000 to 60,000 newborns and is one of the most common genetic causes of childhood epilepsy, of which approximately 90% occur in females. CDKL5 deficiency was previously classified as an atypical form of Rett syndrome, with which they share common features, including seizures, intellectual disability, and other developmental problems. However, patients with CDKL5 deficiency have a significantly different clinical course from Rett syndrome.
  • CDKL5 deficiency In Rett syndrome, seizures generally begin in adolescence and decrease in severity with age; whereas patients with CDKL5 deficiency develop seizures in the first few months of life and are severely resistant to antiepileptic drugs and cannot be cured. Therefore, CDKL5 deficiency is now considered a separate disease from Rett syndrome.
  • CDKL5 deficiency is caused by mutations in the CDKL5 gene. Mutations in the CDKL5 gene reduce the expression level of functional CDKL5 protein or change its activity in nerve cells, but it is not clear how these changes lead to the specific phenotypes of CDKL5 deficiency.
  • the CDKL5 gene is located on the X chromosome and is inherited in an X-linked dominant pattern. Since women have two X chromosomes, random inactivation of the X chromosome can lead to different severity of symptoms and signs in different CDD patients. Women with a higher proportion of mutated neurons have more severe signs and symptoms than women with a lower proportion of mutated neurons. Since men have only one X chromosome in each cell, mutations in the CDKL5 gene are active in all cells, and affected men do not have a normal copy of the gene.
  • Seizures are often the first manifestation of patients with CDKL5 deficiency, with a median time from birth to onset of 4-6 weeks, and more than 90% of patients experience seizures within 3 months of birth, which can occur as early as the first week after birth. Patients' seizures can develop into different types over time and may follow a certain pattern of seizures. The most common types are generalized tonic-clonic seizures, including loss of consciousness, muscle rigidity, and convulsions; tonic seizures, which are characterized by abnormal muscle contractions; and epileptic spasms, in which the EEG shows mild arrhythmias and short-term muscle twitches. Although there may be remission periods (a period of no seizures), most people with CDD may have seizures every day.
  • CDKL5 deficiency will have developmental impairments, most of whom have severe intellectual disabilities, with language skills being particularly affected.
  • the development of gross motor skills such as sitting, standing, and walking is delayed, and only about one-third of CDD patients can walk independently.
  • Fine motor skills (such as picking up small objects with fingers) are also affected, and about half of CDD patients have limited fine motor skills that last a lifetime.
  • Most patients All have vision problems, some of which present as cortical visual impairment (complete loss of binocular vision, normal pupillary light reflex, and normal fundus).
  • Other common features of CDD patients include repetitive hand movements such as clapping, licking, and sucking; teeth grinding; disrupted sleep; feeding difficulties, and gastrointestinal problems, including constipation and gastroesophageal reflux.
  • Some patients have episodic irregular breathing.
  • Other limb abnormalities may also occur, such as head abnormalities (microcephaly), lateral curvature of the spine, and thin fingers.
  • the current medical management of patients with CDKL5 deficiency is mainly symptomatic and supportive treatment, aimed at maximizing the patient's personal abilities and improving any skills that may arise.
  • Emphasis is placed on early intervention treatments such as physical therapy, occupational therapy, and speech and assistive communication therapy.
  • epilepsy control is the most challenging content.
  • the "International Consensus Recommendations for the Evaluation and Management of Patients with CDKL5 Deficiency" points out that existing drugs, including corticosteroids, vigabatrin, valproic acid, phenytoin, felbamate, carbamazepine, clonazepam, oxcarbazepine, and lacosamide, can reduce the frequency of epileptic seizures when used alone in the early stage, but they will lose their efficacy after a certain period of time (median response time is 6 months), and even in some cases, they will aggravate the seizures.
  • drugs including corticosteroids, vigabatrin, valproic acid, phenytoin, felbamate, carbamazepine, clonazepam, oxcarbazepine, and lacosamide
  • the consensus document does not make specific anti-seizure drug treatment recommendations for CDKL5 deficiency, but suggests alternatives, including a ketogenic diet; implantation of a vagus nerve stimulator (VNS, which delivers small pulses of electrical current to the vagus nerve along the neck to the brain); corpus callosotomy (severing the main fibers connecting the two hemispheres of the brain); and pharmaceutical-grade cannabidiol.
  • VNS vagus nerve stimulator
  • corpus callosotomy severed the main fibers connecting the two hemispheres of the brain
  • pharmaceutical-grade cannabidiol cannabidiol.
  • Epilepsy is a central nervous system disease in which patients suffer from recurrent, partial or whole-body seizures, sometimes accompanied by loss of consciousness. It is a catastrophic neurological disease.
  • the severity of seizures in epilepsy patients can range from brief distraction or muscle rigidity to prolonged severe convulsions.
  • Epileptic seizures are generally classified as focal or generalized, depending on how and where the seizure begins.
  • Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy. About 60% of patients with focal epilepsy have temporal lobe epilepsy, which often begins in the hippocampus or limbic system.
  • Temporal lobe epilepsy is considered to be the most suitable for scientific research on epilepsy: as the most common type of epilepsy, temporal lobe epilepsy has a large amount of EEG data and case reports available for research, and because temporal lobectomy has a good therapeutic effect on temporal lobe refractory epilepsy, its pathological tissue is easier to obtain than other types of epilepsy.
  • the most common pathological feature of temporal lobe epilepsy is hippocampal sclerosis, which can be observed in epilepsy patients and animal models. It is mainly manifested by the loss of hippocampal neurons, including the loss of pyramidal neurons in CA1 and CA3, and the diffuse distribution of dentate gyrus cells.
  • the dentate gyrus is a key structure for transmitting excitation to the hippocampus. In epilepsy patients and animal models of epilepsy, the dentate gyrus undergoes a variety of changes, making the hypothesis that epilepsy originates from the hippocampus a hot topic.
  • CDKL5 deficiency is a severe epileptic encephalopathy caused by mutations in the CDKL5 gene. Since CDKL5 deficiency was studied as an independent disease, there have been many research advances on the properties and functions of CDKL5 protein, but these findings cannot clearly explain the symptoms caused by CDKL5 deficiency, and there is no animal model that can well reproduce the spontaneous epileptic phenotype of CDD patients. As the most core clinical symptom, the mechanism of CDD-related spontaneous epilepsy is still unclear.
  • the purpose of the present invention is to provide a new drug for treating epilepsy associated with CDKL5 deficiency.
  • Another object of the present invention is to provide evidence that targeting the BDNF-TrkB signaling pathway can be used to prevent and/or treat CDKL5 deficiency-related epilepsy.
  • the present invention provides a use of a BDNF-TrkB signaling pathway inhibitor for preparing a composition or preparation for preventing and/or treating CDKL5 deficiency-related epilepsy.
  • a first aspect of the present invention provides a use of a BDNF-TrkB signaling pathway inhibitor for preparing a composition or preparation for preventing and/or treating CDKL5 deficiency-related epilepsy.
  • the CDKL5 deficiency-related epilepsy has one or more phenotypic characteristics selected from the following group:
  • the patient's epilepsy phenotype is resistant to antiepileptic drugs
  • EEG electroencephalogram
  • composition or preparation is also used for one or more purposes selected from the following group:
  • the BDNF-TrkB signaling pathway inhibitor includes a TrkB antagonist.
  • the BDNF-TrkB signaling pathway inhibitor is selected from the following group: ANA-12, K252a, or a combination thereof.
  • composition or preparation further comprises other drugs that can prevent and/or treat epilepsy associated with CDKL5 deficiency.
  • other drugs for preventing and/or treating CDKL5 deficiency-related epilepsy include TAK-935/OV935 and Ganaxolone.
  • the composition comprises a pharmaceutical composition.
  • the pharmaceutical composition contains (a) a BDNF-TrkB signaling pathway inhibitor and (b) a pharmaceutically acceptable carrier.
  • the component (a) accounts for 0.1-99.9wt% of the total weight of the pharmaceutical composition, preferably 10-99.9wt%, and more preferably 70%-99.9wt%.
  • the component (a) accounts for 60.0%-99.5wt% of the total weight of the pharmaceutical composition, preferably 70.0-99.5wt%, more preferably 80.0%-99.5wt%.
  • the pharmaceutical composition is liquid, solid, or semisolid.
  • the dosage form of the pharmaceutical composition includes tablets, granules, capsules, oral solutions, or injections.
  • the composition is an oral preparation.
  • composition (such as a pharmaceutical composition) is administered to a mammal by the following method: Drugs: Oral, intravenous, or local injection.
  • the mammal includes a mammal suffering from CDKL5 deficiency-related epilepsy.
  • the mammal includes a human or a non-human mammal.
  • the non-human mammals include rodents, such as mice and rats.
  • the second aspect of the present invention provides a pharmaceutical composition for preventing and/or treating CDKL5 deficiency-related epilepsy, comprising:
  • a first pharmaceutical composition comprising (a) a first active ingredient, the first active ingredient being a BDNF-TrkB signaling pathway inhibitor;
  • the first pharmaceutical composition and the second pharmaceutical composition are different pharmaceutical compositions, or the same pharmaceutical composition.
  • the BDNF-TrkB signaling pathway inhibitor includes a TrkB antagonist.
  • the BDNF-TrkB signaling pathway inhibitor is selected from the following group: ANA-12, K252a, or a combination thereof.
  • other drugs for preventing and/or treating CDKL5 deficiency-related epilepsy include TAK-935/OV935 and Ganaxolone.
  • the weight ratio of the first active ingredient to the second active ingredient is 1:100 to 100:1, preferably 1:10 to 10:1.
  • the content of component (a1) is 1%-99%, preferably, 10%-90%, more preferably, 30%-70%.
  • the component (a1) and the component (a2) account for 0.01-99.99 wt %, preferably 0.1-90 wt %, and more preferably 1-80 wt % of the total weight of the product combination.
  • the dosage form of the pharmaceutical composition includes an injection form and an oral dosage form.
  • the oral dosage form includes tablets, capsules, films, and granules.
  • the dosage form of the pharmaceutical composition includes a sustained-release dosage form and a non-sustained-release dosage form.
  • the weight ratio of component (a1) to component (a2) is 1:100 to 100:1, preferably 1:10 to 10:1.
  • the third aspect of the present invention provides a medicine kit, comprising:
  • first container and the second container are the same or different containers.
  • the drug in the first container is a single-ingredient preparation containing a BDNF-TrkB signaling pathway inhibitor.
  • the drug in the second container is a single-ingredient preparation containing other drugs for preventing and/or treating epilepsy associated with CDKL5 deficiency.
  • the dosage form of the drug is an oral dosage form or an injection dosage form.
  • kit further contains instructions.
  • the description records one or more instructions selected from the following group:
  • the fourth aspect of the present invention provides a use of a combination, wherein the combination comprises a BDNF-TrkB signaling pathway inhibitor and optionally other drugs for preventing and/or treating CDKL5 deficiency-related epilepsy, for preparing a pharmaceutical composition or a kit for preventing and/or treating CDKL5 deficiency-related epilepsy.
  • the combination comprises a BDNF-TrkB signaling pathway inhibitor and optionally other drugs for preventing and/or treating CDKL5 deficiency-related epilepsy, for preparing a pharmaceutical composition or a kit for preventing and/or treating CDKL5 deficiency-related epilepsy.
  • the pharmaceutical composition or kit comprises (a) a BDNF-TrkB signaling pathway inhibitor and optionally other drugs for preventing and/or treating CDKL5 deficiency-related epilepsy; and (b) a pharmaceutically acceptable carrier.
  • the BDNF-TrkB signaling pathway inhibitor and the drug for preventing and/or treating CDKL5 deficiency-related epilepsy respectively account for 0.01-99.99wt%, preferably 0.1-90wt%, and more preferably 1-80wt% of the total weight of the pharmaceutical composition or medicine kit.
  • a fifth aspect of the present invention provides a method for preventing and/or treating epilepsy associated with CDKL5 deficiency, comprising:
  • a BDNF-TrkB signaling pathway inhibitor and optionally other drugs for preventing and/or treating CDKL5 deficiency-related epilepsy, or the pharmaceutical composition of the second aspect of the present invention or the drug kit of the third aspect of the present invention is administered to a subject in need thereof.
  • the subject includes a human or non-human mammal suffering from CDKL5 deficiency-related epilepsy.
  • the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.
  • Figure 1 shows the domains of CDKL5 protein and pathogenic mutations.
  • the functional domains of CDKL5 protein are marked with different colors, and the numbers refer to the type and position of amino acids. Red marks are pathogenic or potentially pathogenic mutations; black marks are benign or potentially benign mutations.
  • Figure 2 shows a neural circuit diagram of the rodent hippocampus.
  • A, B A neural circuit diagram of the rodent hippocampus and a schematic diagram of the corresponding neural network.
  • the solid arrows depict the neural circuit of the entorhinal cortex (EC)-dentate gyrus-CA3-CA1-EC.
  • Neurons in layer II of the entorhinal cortex travel to the dentate gyrus through the perforated path (PP).
  • the dentate gyrus projects axons, including the lateral perforant pathway (LPP) and the medial perforant pathway (MPP).
  • the dentate gyrus projects to the pyramidal cells of CA3 via mossy fibers.
  • CA3 pyramidal neurons transmit information to CA1 pyramidal neurons via Schaffer collaterals.
  • CA1 pyramidal neurons output to the deep neurons of the entorhinal cortex.
  • CA3 also receives direct projections from the entorhinal cortex via the perforant pathway.
  • CA1 receives direct input from the entorhinal cortex via the temporoamnion pathway (TA).
  • TA temporoamnion pathway
  • Dentate granule cells also project to the mossy cells in the hilus region and the interneurons in the hilar region, and project back to the granule cells, respectively.
  • Figure 3 shows the biological actions of BDNF.
  • Na + influx depolarizes the membrane, which triggers an influx of Ca2 + and the release of the excitatory neurotransmitter glutamate into the synaptic cleft.
  • Glutamate binds to AMPA and NMDA receptors on the postsynaptic membrane. Activation of the receptors leads to membrane depolarization and influx of Ca2 + via NMDA and VDCC.
  • Ca2 + binds to CaMKs that activate CREB and NF-kB, which in turn induce transcription of the Bdnf gene.
  • BDNF is released at the synaptic cleft and activates the TrkB receptor, leading to the activation of downstream signaling cascades including PLC ⁇ , PI3K, and MAPKs and the subsequent expression of genes that are critical for neuronal survival and plasticity.
  • BDNF signaling also has acute effects on membrane excitability and synaptic transmission by altering the kinetics of NMDA receptors and increasing the number of synaptic vesicles presynaptically.
  • FIG. 4 shows that Cdk15 fl/Y ;Emx1-Cre mice develop spontaneous epilepsy phenotype.
  • FIG. B Representative images of electroencephalogram (EEG) and electromyogram (EMG) recordings during spontaneous epileptic seizures in Cdkl5 fl/Y ; Emx1-Cre mice.
  • the target mice were 6 months old.
  • the upper and middle images show the changes in electroencephalogram (EEG) recordings during epileptic seizures in Cdkl5 fl/Y ;Emx1-Cre mice.
  • the top arrow represents the onset of epileptic seizures.
  • the amplitude of epileptic seizures increased significantly during epileptic seizures, and there was suppression after the end of the seizures.
  • the lower image shows the changes in electromyogram (EMG) recordings during epileptic seizures in Cdkl5 fl/Y ;Emx1-Cre mice.
  • C Statistical graph of spontaneous epileptic seizure rates in Cdkl5 fl/Y ;Emx1-Cre mice and their control group. There were 10 Cdkl5 fl/Y mice and 15 Cdkl5 fl/Y ;Emx1-Cre mice.
  • FIG. 5 shows that Cdk15 fl/Y ;CaMK2 ⁇ -iCre mice develop spontaneous epilepsy phenotype.
  • FIG. 1 Representative images of electroencephalogram (EEG) and electromyogram (EMG) recordings during spontaneous epileptic seizures in Cdkl5 fl /Y ;CaMK2 ⁇ -iCre mice.
  • the target mice were 6 months old.
  • the upper figure shows the changes in electroencephalogram (EEG) recordings during epileptic seizures in Cdkl5 fl/Y ;CaMK2 ⁇ -iCre mice.
  • the top arrow represents the onset of epileptic seizures.
  • the amplitude of epileptic seizures increased significantly during epileptic seizures and was suppressed after the end.
  • the lower figure shows the changes in electromyogram (EMG) recordings during epileptic seizures in Cdkl5 fl/Y ;CaMK2 ⁇ -iCre mice.
  • C Statistical graph of spontaneous epileptic seizure rates in Cdkl5 fl/Y ;CaMK2 ⁇ -iCre mice and their control group. There were 12 Cdkl5 fl/Y mice and 15 Cdkl5 fl/Y ;CaMK2 ⁇ -iCre mice.
  • FIG6 shows that excitatory transmission in the dentate gyrus of the hippocampus of Cdk15 fl/Y ;Emx1-Cre mice is enhanced.
  • FIG. 7 shows that excitatory transmission in the dentate gyrus of the hippocampus of Cdk15 fl/Y ;CaMK2 ⁇ -iCre mice is enhanced.
  • FIG8 shows that the amplitude of mIPSCs in the dentate gyrus of the hippocampus of Cdkl5 fl/Y ;Emx1-Cre mice is enhanced.
  • FIG. 9 shows that Cdk15 fl/Y ;CaMK2 ⁇ -CreER mice develop spontaneous epilepsy.
  • FIG. 10 shows that excitatory transmission in the dentate gyrus of the hippocampus of Cdk15 fl/Y ;CaMK2 ⁇ -CreER mice is enhanced.
  • the control group consisted of 3 mice in the Cdkl5 fl/Y + tamoxifen group, with 11 neurons recorded; the Cdkl5 fl /Y ;CaMK2 ⁇ -CreER + corn oil group consisted of 3 mice, with 14 neurons recorded; the experimental group consisted of 3 mice in the Cdkl5 fl/Y ;CaMK2 ⁇ -CreER + tamoxifen group, with 11 neurons recorded. ****p ⁇ 0.0001, unpaired two-tailed t-test.
  • FIG. 11 shows that the density and maturity of dendritic spines in granule cells of Cdk15 fl/Y ;CaMK2 ⁇ -CreER mice are normal.
  • C Classification of dendritic spines of hippocampal dentate gyrus granule cells in Cdkl5 fl/Y ;CaMK2 ⁇ -CreER+tamoxifen mice and their control group. Two-way ANOVA with Bonferroni post hoc test. Each group contained 41-52 neurons.
  • FIG12 shows that the BDNF expression level in the hippocampus of Cdkl5 fl/Y ;CaMK2 ⁇ -CreER mice is increased, and the BDNF-TrkB signaling pathway is abnormally activated.
  • C qPCR analysis of BDNF mRNA levels in the hippocampus of Cdkl5 fl/Y ; CaMK2 ⁇ -CreER mice and their control group.
  • the control group consisted of 11 mice in the Cdkl5 fl/Y + tamoxifen group; 5 mice in the Cdkl5 fl / Y ; CaMK2 ⁇ -CreER + corn oil group; and the experimental group consisted of 10 mice in the Cdkl5 fl/Y ; CaMK2 ⁇ -CreER + tamoxifen group.
  • the control group consisted of 14 mice in the Cdkl5 fl/Y + tamoxifen group, 8 mice in the Cdkl5 fl /Y ; CaMK2 ⁇ -CreER + corn oil group, and the experimental group consisted of 12 mice in the Cdkl5 fl/Y ; CaMK2 ⁇ -CreER + tamoxifen group.
  • FIG. 13 shows that the Trk inhibitor K252a can improve the defect of enhanced excitatory synaptic transmission in Cdk15 fl/Y ;CaMK2 ⁇ -CreER mice.
  • the control group consisted of 4 mice in the Cdkl5 fl/Y + tamoxifen group, from which 11 neurons were recorded; the Cdkl5 fl/Y ;CaMK2 ⁇ CreER+corn oil group consisted of 3 mice, from which 10 neurons were recorded; the experimental group consisted of 4 mice in the Cdkl5 fl/Y ;CaMK2 ⁇ -CreER+tamoxifen group, from which 13 neurons were recorded.
  • FIG. 14 shows that the TrkB antagonist ANA-12 can improve the defect of enhanced excitatory synaptic transmission in Cdk15 fl/Y ;CaMK2 ⁇ -CreER mice.
  • FIG. 15 shows that knocking down TrkB receptor can improve spontaneous epileptic activity caused by knocking out Cdk15 in adulthood.
  • FIG. 16 shows that inhibition of TrkB receptor can improve spontaneous epileptic activity in Cdk15 fl/Y ;CaMK2 ⁇ -CreER mice.
  • BDNF-TrkB signaling pathway inhibitors such as TrkB antagonists
  • TrkB antagonists can effectively prevent and/or treat CDKL5 deficiency-related epilepsy.
  • ANA-12 has the structural formula:
  • K252a has the structural formula:
  • Ganaxolone As used herein, the Chinese name of the term “Ganaxolone” is Ganaxolone, and the structural formula is:
  • CDKL5 deficiency disorder is a rare genetic disease characterized by seizures that begin in infancy and are accompanied by significant developmental delays in multiple systems. CDKL5 deficiency occurs in approximately 1 in 40,000 to 60,000 newborns and is one of the most common genetic causes of childhood epilepsy, of which approximately 90% occur in females. CDKL5 deficiency was previously classified as an atypical form of Rett syndrome, with which they share common features, including seizures, intellectual disability, and other developmental problems. However, patients with CDKL5 deficiency have a significantly different clinical course from Rett syndrome.
  • CDKL5 deficiency In Rett syndrome, seizures generally begin in adolescence and decrease in severity with age; whereas patients with CDKL5 deficiency develop seizures in the first few months of life and are severely resistant to antiepileptic drugs and cannot be cured. Therefore, CDKL5 deficiency is now considered a separate disease from Rett syndrome.
  • CDKL5 deficiency is caused by mutations in the CDKL5 gene. Mutations in the CDKL5 gene reduce the expression level of functional CDKL5 protein or change its activity in nerve cells, but it is not clear how these changes lead to the specific phenotypes of CDKL5 deficiency.
  • the CDKL5 gene is located on the X chromosome and is inherited in an X-linked dominant pattern. Since women have two X chromosomes, random inactivation of the X chromosome can lead to different severity of symptoms and signs in different CDD patients. Women with a higher proportion of mutated neurons have more severe symptoms than women with a lower proportion of mutated neurons. Because males have only one X chromosome in each cell, mutations in the CDKL5 gene are active in all cells, and affected males do not have a normal copy of the gene.
  • Seizures are often the first manifestation of patients with CDKL5 deficiency, with a median time from birth to onset of 4-6 weeks, and more than 90% of patients experience seizures within 3 months of birth, which can occur as early as the first week after birth. Patients' seizures can develop into different types over time and may follow a certain pattern of seizures. The most common types are generalized tonic-clonic seizures, including loss of consciousness, muscle rigidity, and convulsions; tonic seizures, which are characterized by abnormal muscle contractions; and epileptic spasms, in which the EEG shows mild arrhythmias and short-term muscle twitches. Although there may be remission periods (a period of no seizures), most people with CDD may have seizures every day.
  • CDKL5 deficiency will have developmental impairments, most of whom have severe intellectual disabilities, with language skills being particularly affected.
  • the development of gross motor skills such as sitting, standing, and walking is delayed, and only about one-third of CDD patients can walk independently.
  • Fine motor skills (such as picking up small objects with fingers) are also affected, and about half of CDD patients have limited fine motor skills and will accompany them for life.
  • Most patients have vision problems, some of which manifest as cortical visual impairment (complete loss of binocular vision, normal pupil light reflex, and normal fundus).
  • Other common features of CDD patients include repetitive hand movements such as clapping, licking, and sucking; teeth grinding; interrupted sleep; feeding difficulties and gastrointestinal problems, including constipation and gastroesophageal reflux.
  • Some patients have paroxysmal irregular breathing.
  • Other limb abnormalities may also occur, such as head abnormalities (microcephaly), lateral curvature of the spine, and thin fingers.
  • the current medical management of patients with CDKL5 deficiency is mainly symptomatic and supportive treatment, aimed at maximizing the patient's personal abilities and improving any skills that may arise.
  • Emphasis is placed on early intervention treatments such as physical therapy, occupational therapy, and speech and assistive communication therapy.
  • epilepsy control is the most challenging content.
  • the "International Consensus Recommendations for the Evaluation and Management of Patients with CDKL5 Deficiency" points out that existing drugs, including corticosteroids, vigabatrin, valproic acid, phenytoin, felbamate, carbamazepine, clonazepam, oxcarbazepine, and lacosamide, can reduce the frequency of epileptic seizures when used alone in the early stage, but they will lose their efficacy after a certain period of time (median response time is 6 months), and even in some cases, they will aggravate the seizures.
  • drugs including corticosteroids, vigabatrin, valproic acid, phenytoin, felbamate, carbamazepine, clonazepam, oxcarbazepine, and lacosamide
  • the consensus document does not make specific anti-seizure drug treatment recommendations for CDKL5 deficiency, but suggests alternatives, including a ketogenic diet; implantation of a vagus nerve stimulator (VNS, which delivers small pulses of electrical current to the vagus nerve along the neck to the brain); corpus callosotomy (severing the main fibers connecting the two hemispheres of the brain); and pharmaceutical-grade cannabidiol.
  • VNS vagus nerve stimulator
  • corpus callosotomy severed the main fibers connecting the two hemispheres of the brain
  • pharmaceutical-grade cannabidiol cannabidiol.
  • CDKL5 Cyclin-dependent Kinase Like 5
  • STK9 Cyclin-dependent Kinase Like 5
  • the CDKL5 gene is located in region 22 of the short arm of chromosome X (Xp22).
  • the human CDKL5 gene has 24 exons.
  • the N-terminus of CDKL5 protein is a serine/threonine kinase domain.
  • the pathogenic mutations are most common in the catalytic region ( Figure 1). Exons 2-21 are the coding region.
  • CDKL5 protein belongs to the cell cycle dependent kinase family and is highly homological with the kinase domains of cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAPKs), and glycogen synthase kinases (GSKs).
  • CDKs cyclin-dependent kinases
  • MAPKs mitogen-activated protein kinases
  • GSKs glycogen synthase kinases
  • CDKL5 protein is expressed in large quantities in the central nervous system of humans and rodents, among which the highest expression levels are in the cerebral cortex, hippocampus, thalamus, striatum and olfactory bulb. CDKL5 is expressed at a low level in the embryonic period, and the expression level increases significantly after birth, reaching a peak in the first few weeks, and maintaining stable expression in adulthood. Neurons are the main cell type expressing CDKL5, and glial cells only express a small amount. CDKL5 is expressed in various subcellular structures, among which the cytoplasm is the main expression area, but nuclear aggregation gradually appears as the brain develops.
  • CDKL5 Some potential substrates of CDKL5 have been identified, and some of the substrates related to physiological functions have been verified in cells.
  • substrates include MAP1S, EB2, CEP131, a regulator of microtubule and centrosome function, and ELOA involved in DNA damage response, etc.
  • CDKL5 is mainly enriched in the nucleus in the area of RNA splicing and processing. CDKL5 also accumulates in different subcellular regions at different developmental stages: in the early stages of cultured neurons, CDKL5 was found to accumulate in growth cones; in mature neurons, CDKL5 accumulates in dendritic spines, especially in the postsynaptic density; some studies have also found that CDKL5 exists in centrosomes. These expression patterns indicate that CDKL5 is involved in the regulation of neuronal development and plays a role in the maturation of adult neurons.
  • CDKL5 plays different functions at different stages of neuronal development. In early neuronal development, CDKL5 regulates cell proliferation and neuronal migration. According to previous studies, CDKL5 has a negative regulatory function in regulating cell proliferation. Upregulating CDKL5 expression inhibits the proliferation of human neuroblastoma cells; while knocking out mouse Cdkl5 increases the proliferation rate of dentate gyrus granule cells. In addition, CDKL5 was found to be present in the centrosomes of dividing cells and post-mitotic neurons, indicating that it may regulate cell proliferation and division by affecting the centrosome. CDKL5 has a regulatory effect on neuronal migration, and it was found to interact with IQGAP1, an important regulator related to cell migration and polarity.
  • IQGAP1 an important regulator related to cell migration and polarity.
  • CDKL5 In rodent model animals, reducing the expression of CDKL5 in neural progenitor cells leads to delayed migration of layer 2-3 pyramidal neurons. In the process of neuronal maturation, CDKL5 is closely related to the growth and development of dendrites and axons, and is one of the key proteins for synapse formation. CDKL5 interacts with Shootin1, a key protein for axon growth, in growth cones.
  • CDKL5 Silencing Cdkl5 by RNAi or overexpressing CDKL5 leads to an increase in the number of neurons with multiple axons, but downregulating Cdkl5 does not hinder axon formation, indicating that CDKL5 has a regulatory function in axon growth, but does not play a decisive role in axon formation.
  • CDKL5 also regulates dendrite development. When Cdkl5 is silenced in cultured neurons, the dendritic branching of neurons is severely impaired. Cdkl5 knockout mice can be observed to have a significant reduction in the total length of dendrites in the cerebral cortex and hippocampal CA1 pyramidal neurons, as well as dentate gyrus granule cells.
  • CDKL5 Overexpression of CDKL5 in cultured neurons results in a kinase activity-dependent increase in total dendrite length. In addition, CDKL5 is also involved in synapse formation. CDKL5 has been observed to interact with palmitoylated PSD95 and the adhesion molecule NGL1. PSD is a major scaffolding protein that regulates the localization of synaptic proteins and synaptic strength; NGL1 is a key adhesion molecule for synapse formation. In dendritic spines, CDKL5 is highly enriched in the postsynaptic density, a dense protein complex composed of key proteins for synaptic transmission, signal transduction, and cell adhesion. Synaptic localization strongly suggests that this protein is involved in synaptic development and function.
  • CDKL5 The function of CDKL5 is not limited to neuronal development, and its expression persists in the adult brain. In adult mice lacking CDKL5, the stability and long-term potentiation of mature dendritic spines are impaired, and these animals have defects in hippocampal-dependent memory. Notably, these defects can be reversed by restoring CDKL5 protein levels in adult mice, including abnormal morphology of dendritic spines, hindlimb grasping, and learning and memory abilities. The defects of CDKL5 knockout mice can also be improved by activating the downstream signaling pathways of CDKL5. For example, the application of IGF-1 can restore the stability of dendritic spines, indicating that CDKL5 also plays an important role in the neural maturation period after adulthood.
  • CDKL5 itself are also regulated by neural activity, such as DYRK1A phosphorylation of serine residue 308 promotes the cytoplasmic localization of CDKL5 in Neuro-2a cells; in cultured neurons, BDNF induces transient phosphorylation of CDKL5; neuronal activity prompts PP1 to dephosphorylate CDKL5.
  • neural activity such as DYRK1A phosphorylation of serine residue 308 promotes the cytoplasmic localization of CDKL5 in Neuro-2a cells; in cultured neurons, BDNF induces transient phosphorylation of CDKL5; neuronal activity prompts PP1 to dephosphorylate CDKL5.
  • neural activity such as DYRK1A phosphorylation of serine residue 308 promotes the cytoplasmic localization of CDKL5 in Neuro-2a cells; in cultured neurons, BDNF induces transient phosphorylation of CDKL5; neuronal activity prompts PP1 to dephosphorylate CDKL
  • Cdkl5 exon knockout mice are the most commonly used Cdkl5 knockout mouse model. In this strain of Cdkl5 knockout mice, researchers observed impairment of limb coordination, motor skills, learning and memory abilities, autism-like symptoms, abnormal eye movements, abnormal breathing and sleep patterns, and atypical behavioral responses to whisker-mediated tactile stimulation. Most of these phenotypes can be mutually confirmed with the symptoms of patients with CDKL5 deficiency.
  • CDKL5 deficiency mouse model confirmed the importance of this gene for normal brain development and nervous system function, and also provided an in vivo environment to discover and validate various protein-protein interactions and signaling pathways involving CDKL5, and to test newly discovered treatments.
  • CDKL5 the endogenous function of CDKL5 and its role in the development, maintenance, and pathogenesis of the nervous system remain to be elucidated, and the core spontaneous epilepsy phenotype of CDKL5 deficiency has not been well reproduced, and there is still a lack of a new animal model to simulate CDD-related epilepsy.
  • Epilepsy is a central nervous system disease in which patients suffer from recurrent seizures involving part or all of the body, sometimes accompanied by loss of consciousness. It is a catastrophic neurological disease. The severity of seizures in epilepsy patients can range from brief distractions or muscle rigidity to prolonged severe convulsions. Epileptic seizures are generally classified as focal or generalized, depending on how and where the seizure begins. Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy. Approximately 60% of patients with focal epilepsy have temporal lobe epilepsy, which often begins in the hippocampus or limbic system.
  • Temporal lobe epilepsy is considered the most suitable for scientific research on epilepsy: as the most common type of epilepsy, temporal lobe epilepsy has a large amount of EEG data and case reports available for research, and because temporal lobectomy has a good therapeutic effect on refractory temporal lobe epilepsy, it ... Pathological tissue is easier to obtain than other types of epilepsy.
  • the most common pathological feature of temporal lobe epilepsy is hippocampal sclerosis, which can be observed in both epilepsy patients and animal models. It is mainly manifested by the loss of hippocampal neurons, including the loss of pyramidal neurons in CA1 and CA3, and the diffuse distribution of dentate gyrus cells.
  • the dentate gyrus is a key structure for the transmission of excitement to the hippocampus. In epilepsy patients and animal models of epilepsy, the dentate gyrus undergoes a variety of changes, making the hypothesis that epilepsy originates from the hippocampus a research hotspot.
  • the hippocampus is located in the medial region of the temporal lobe and plays an important role in emotion regulation, consolidation of information from short-term to long-term memory, and spatial processing. Understanding the anatomy of the hippocampus is crucial to its cognitive function.
  • the hippocampus consists of subregions such as Cornu ammonis (CA, including CA1-CA4), dentate gyrus and subthalamic region. Axons of neurons in layer II of the entorhinal cortex project to the dentate gyrus through the perforant pathway (PP), including the lateral perforant pathway (LPP) and the medial perforant pathway (MPP).
  • PP perforant pathway
  • LPP lateral perforant pathway
  • MPP medial perforant pathway
  • the dentate gyrus projects to the pyramidal cells of CA3 through mossy fibers, which are then transmitted to CA1 pyramidal neurons through Schaffer collaterals, and finally output to the deep neurons of EC through CA1 pyramidal neurons.
  • CA3 also receives direct projections from the entorhinal cortex through the perforant pathway; CA1 receives direct input from the entorhinal cortex through the temporo-ammonic pathway (TA).
  • TA temporo-ammonic pathway
  • the dentate granule cells project to interneurons and mossy cells, and receive their inhibitory and excitatory feedback respectively ( Figure 2, A-B).
  • the dentate gyrus consists of three layers from outside to inside: the molecular layer, the granule cell layer, and the polymorphic layer (also called the Hilus layer).
  • Granule cells as the main neurons of the dentate gyrus, are densely arranged in the granule cell layer, and their axons project to CA3 to form excitatory synapses with pyramidal neurons.
  • the molecular layer is a cell-free layer occupied by the dendrites of granule cells in the granule cell layer and the perforated path fibers from the entorhinal cortex.
  • mossy cells are one of the most important neurons.
  • the granular layer and the polymorphic layer there are several types of neurons, such as pyramidal basket cells, which are inhibitory interneurons.
  • neurons such as pyramidal basket cells, which are inhibitory interneurons.
  • the mossy fibers projected by granule cells terminate above the CA3 pyramidal cell layer, which is called the clear layer.
  • Mossy fibers also project to the polymorphic layer to form synaptic connections with interneurons.
  • the activity of granule cells in the dentate gyrus is very low, which is related to both the intrinsic characteristics of their own cells and the circuit network environment in which they are located.
  • granule cells exhibit a hyperpolarized resting membrane potential, which makes them more difficult to induce action potentials.
  • granule cell dendrites show significant voltage-dependent linear integration and strong attenuation of synaptic inputs to the entorhinal cortex.
  • dentate gyrus granule cells are also in a circuit network that is biased towards inhibition.
  • feedforward inhibition is provided by a variety of inhibitory neurons such as fast-spiking interneurons, hilar interneurons, and basket cells.
  • Granule cell axonal projections are also connected to a variety of GABAergic interneurons, which form dendritic targeted feedback regulation on granule cells.
  • the intrinsic properties of granule cells and the inhibitory circuit network environment make it exist as a shock absorber between the entorhinal cortex and CA3. Therefore, when abnormalities occur in the dentate gyrus, it is considered to be the "detonator/detonator" of the epileptogenic process, and together with CA3, it becomes the key to the formation of epileptic seizures, which is also called the gating hypothesis of the dentate gyrus.
  • epilepsy studies that support this theory and explain the gating hypothesis of the dentate gyrus, including mossy fiber sprouting (abnormal synapses formed on mossy fibers and other granule cells); abnormal formation and persistence of basal dendrites of granule cells; ectopic dispersion and migration of granule cells; changes in the intrinsic properties of granule cell membranes and synaptic receptor expression; downregulation of inhibitory GBAB synaptic transmission and abnormal regulation of various molecular signaling pathways.
  • the use of optogenetics to depolarize or hyperpolarize granule cells in animal models can effectively inhibit/induce the occurrence of spontaneous epilepsy, proving that the dentate gyrus is a key node in the temporal lobe epilepsy seizure network.
  • Optogenetic activation of inhibitory interneurons in the dentate gyrus can prevent the spread of epilepsy in the hippocampus and cortex, further highlighting the important role of the dentate gyrus in regulating cortical input.
  • CDD patients with CDKL5 deficiency will show different types of epileptic seizures at different stages of the disease course, but CDD patients can be observed to have high-intensity temporal lobe activity in MRI, and multiple case reports have shown temporal lobe atrophy in CDD patients; common pathological features of temporal lobe epilepsy such as mossy fiber sprouting can also be seen in CDD animal models, and these evidences indicate that CDD-related epilepsy is closely related to temporal lobe epilepsy. In CDD mouse models, various changes in the dentate gyrus of the hippocampus can also be observed.
  • the molecular layer of the dentate gyrus has reduced immunoreactivity to presynaptic markers synaptophysin and VGLUT1, indicating impaired synaptic maintenance;
  • GluN2B subunits accumulate ectopically at the synaptic junctions of the dentate gyrus granule cells-CA3 layer, causing synaptic hyperexcitation and increased susceptibility to epilepsy;
  • the death of postmitotic granule neuron precursors increases, the total number of granule cells decreases, and the newly generated granule cells show severe dendritic atrophy, which impairs hippocampal-dependent behaviors;
  • the density of dendritic spines and the number of mature dendritic spines of dentate gyrus granule cells and CA1 pyramidal neurons are significantly reduced.
  • Brain derived neurotrophic factor belongs to the neurotrophic factor family and is related to the typical nerve growth factor (NGF). The family also includes NT-3 and NT-4/NT5. BDNF acts on certain neurons in the central and peripheral nervous systems. In the brain, it is active in the hippocampus, cortex, and basal forebrain regions. It is also expressed in the retina, kidney, prostate, motor neurons, and skeletal muscle. BDNF mainly binds to the TrkB receptor with high affinity. The low-affinity nerve growth factor receptor LNGFR (also known as p75) can also respond to BDNF. BDNF plays different roles in neurons at different time and space stages. The existence of complex multi-level regulation proves the importance and diversity of BDNF functions.
  • NGF nerve growth factor
  • BDNF plays a vital role in the survival and differentiation of neuronal populations, promoting the growth and differentiation of newborn neurons by changing cell survival and proliferation; BDNF plays a key role in regulating plastic changes in the adult brain, including regulating protein transport, receptor phosphorylation, and glutamate receptor expression levels; BDNF can promote changes in the morphology of dendritic spines, increase the number, size, and complexity of dendritic spines, thereby stabilizing long-term potentiation (LTP), and plays a key role in memory formation and maintenance.
  • LTP long-term potentiation
  • BDNF-TrkB signaling pathway is abnormally upregulated during epilepsy, and this upregulation is closely related to the excitatory/inhibitory homeostasis of the neural circuit.
  • the gyrus is a key structure in the epileptogenic effect of BDNF. Increased BDNF expression levels may not only lead to structural reorganization of the hippocampal circuit (mossy fiber sprouting), but also affect synaptic transmission in multiple regions of the hippocampus (including enhanced glutamate-mediated excitatory synaptic transmission or GABA-mediated inhibitory synaptic transmission). These changes can lead to a hyperexcitable state of the hippocampal circuit, further promoting abnormal activation of BDNF, thereby forming status epilepticus.
  • CDKL5 deficiency-associated epilepsy disorder refers to an epilepsy phenotype caused by CDKL5 deficiency.
  • the CDKL5 deficiency-related epilepsy disease has one or more phenotypic characteristics selected from the following group:
  • the patient's epilepsy phenotype is resistant to antiepileptic drugs.
  • EEG electroencephalogram
  • the present invention focuses on exploring the pathogenesis of epilepsy associated with CDKL5 deficiency. We found that knocking out Cdkl5 in forebrain excitatory neurons can cause mice to have spontaneous epileptic phenotypes, and the excitatory synaptic transmission in the dentate gyrus of the hippocampus is enhanced, whether in the developmental period or in adulthood.
  • the density and maturity of dendritic spines of granule cells in the dentate gyrus were normal, and the electrophysiological properties mediated by AMPA receptors were not affected, but the BDNF-TrkB signaling pathway in the hippocampus was abnormally upregulated.
  • BDNF-TrkB signaling pathway By inhibiting the BDNF-TrkB signaling pathway, abnormal synaptic transmission in the dentate gyrus can be rescued and epileptic activity in Cdkl5 fl/Y ;CaMK2 ⁇ -CreER mice can be reduced, suggesting that abnormal upregulation of the BDNF-TrkB signaling pathway promotes epilepsy in genetic epilepsy mouse models, and targeting this pathway may be an effective strategy for treating CDD-related epilepsy.
  • the BDNF-TrkB signaling pathway inhibitor refers to a small molecule that can inhibit the TrkB activity induced by BDNF in a competitive or non-competitive manner, including TrkB antagonists.
  • the BDNF-TrkB signaling pathway inhibitor is selected from the group consisting of ANA-12, K252a, or a combination thereof.
  • the present invention finds for the first time that BDNF-TrkB signaling pathway inhibitors can effectively prevent and/or treat CDKL5 deficiency-related epilepsy.
  • the present invention provides a composition comprising active ingredients (a) a BDNF-TrkB signaling pathway inhibitor; optionally (b) a drug for preventing and/or treating epilepsy associated with CDKL5 deficiency; and (c) a pharmaceutically acceptable carrier.
  • Compound pharmaceutical composition include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, powders, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods.
  • compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions.
  • the pharmaceutical combination of the present invention can also be prepared in powder form for aerosol inhalation.
  • a preferred dosage form is an injection preparation.
  • the pharmaceutical composition of the present invention can also be used with other therapeutic agents.
  • the present invention also provides a drug kit for preventing and/or treating CDKL5 deficiency-related epilepsy, the drug kit comprising:
  • (b1) an optional second container, and other drugs for preventing and/or treating epilepsy associated with CDKL5 deficiency located in the second container, or containing other drugs for preventing and/or treating epilepsy associated with CDKL5 deficiency.
  • compositions and kit of the present invention are suitable for preventing and/or treating epilepsy associated with CDKL5 deficiency.
  • the preparation of the present invention can be taken three times a day to once every ten days, or once every ten days in a sustained-release manner.
  • the preferred mode is to take it once a day, because it is convenient for patients to adhere to it, thereby significantly improving the compliance of patients to take the medicine.
  • the total daily dose in most cases should be lower than (or equal to or slightly higher than) the daily dose of each single drug in a few cases.
  • the effective dose of the active ingredient used may vary depending on the mode of administration and the severity of the disease to be treated.
  • the present invention also provides a method for preventing and/or treating CDKL5 deficiency-related epilepsy using the above-mentioned active ingredients or corresponding drugs of the present invention, which comprises administering to a mammal an effective amount of the active ingredient (a) a BDNF-TrkB signaling pathway inhibitor; optionally (b) other drugs for preventing and/or treating CDKL5 deficiency-related epilepsy (such as TAK-935/OV935 and Ganaxolone), or administering a pharmaceutical composition containing the active ingredient (a) and the optional active ingredient (b).
  • a BDNF-TrkB signaling pathway inhibitor optionally
  • other drugs for preventing and/or treating CDKL5 deficiency-related epilepsy such as TAK-935/OV935 and Ganaxolone
  • the active ingredient of the present invention can be mixed with one or more pharmaceutically acceptable carriers or excipients, such as solvents, diluents, etc., and can be administered orally in the form of tablets, pills, capsules, dispersible powders, granules or suspensions (containing, for example, about 0.05-5% suspending agents), syrups (containing, for example, about 10-50% sugar), and elixirs (containing about 20-50% ethanol), or parenterally in the form of sterile injectable solutions or suspensions (containing about 0.05-5% suspending agents in isotonic media).
  • these pharmaceutical preparations can contain about 0.01-99%, more preferably about 0.1%-90% (weight) of the active ingredient mixed with a carrier.
  • the active ingredients or pharmaceutical compositions of the present invention can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, oral, intratumoral or topical administration.
  • routes of administration include oral administration, intramuscular administration or intravenous administration.
  • the preferred pharmaceutical composition is a liquid composition, especially an injection.
  • BDNF-TrkB signaling pathway inhibitors such as TrkB antagonists
  • TrkB antagonists can Effectively prevent and/or treat CDKL5 deficiency-related epilepsy.
  • the present invention focuses on exploring the mechanism of epilepsy associated with CDKL5 deficiency. We found that knocking out Cdkl5 in forebrain excitatory neurons can cause mice to have spontaneous epilepsy phenotypes, and the excitatory synaptic transmission in the dentate gyrus of the hippocampus is enhanced , whether in the developmental period or in adulthood.
  • BDNF-TrkB signaling pathway By inhibiting the BDNF-TrkB signaling pathway, abnormal synaptic transmission in the dentate gyrus can be rescued and epileptic activity in Cdkl5 fl/Y ;CaMK2 ⁇ -CreER mice can be reduced, suggesting that abnormal upregulation of the BDNF-TrkB signaling pathway promotes epilepsy in genetic epilepsy mouse models, and targeting this pathway may be an effective strategy for treating CDD-related epilepsy.
  • the present invention first discovered that previous CDD animal models could not well replicate the core epilepsy phenotype of CDD.
  • the present invention constructed a mouse model with a spontaneous epilepsy phenotype by knocking out Cdkl5 in the excitatory neurons of the forebrain.
  • CDKL5 maintains the stability of the hippocampal neural network in adulthood, illustrating that CDKL5 deficiency is not only a neurodevelopmental disorder but also a neurofunctional disorder.
  • the present invention observed for the first time that the BDNF-TrkB signaling pathway was abnormally upregulated in adult Cdkl5 knockout mice, and that inhibition of the BDNF-TrkB signaling pathway could reduce spontaneous epileptic seizure activity, thus proposing a molecular mechanism for the occurrence of CDD-related epilepsy.
  • the research of the present invention illustrates the important role of CDKL5 in the balance of excitatory/inhibitory synaptic transmission, and provides a new model and new target for the occurrence of CDD-related epilepsy, laying a more solid foundation for the clinical treatment of CDD-related epilepsy.
  • the reagents related to mouse experiments are as follows:
  • Tamoxifen formulation (final concentration: 100 mg/kg): 0.5 g of tamoxifen (MedChemExpress, 1052910) was dissolved in a mixture of 2.5 ml of anhydrous ethanol (Sinopharm, 10009228) and 47.5 ml of corn oil (ABCONE, C67366), mixed in a 40°C water bath until completely dissolved, and stored in aliquots at -20°C. 0.1 ml was injected for every 10 g of mouse body weight.
  • ANA-12 formulation (final concentration: 6 mg/kg): Dissolve 1.2 mg ANA12 (MedChemExpress, HY-12497) in 100 ⁇ l dimethyl sulfoxide (DMSO) solution (Sigma-Aldrich, D2447), add 900 ⁇ l corn oil (ABCONE, C67366), shake until no precipitation, store at -20°C, and use within one week. Inject 0.1 ml for every 20 g of mouse body weight.
  • DMSO dimethyl sulfoxide
  • VPA formula final concentration: 50 mg/kg: 10 mg valproic acid (Valproic Acid, TargetMol, T7064) was dissolved in 100 ⁇ l dimethyl sulfoxide (DMSO) solution (Sigma-Aldrich, D2447), and then 900 ⁇ l corn oil (ABCONE, C67366) was added. The mixture was shaken until there was no precipitation, and stored at -20°C. It was used within one week. 0.1 ml was injected for every 20 g of mouse body weight.
  • DMSO dimethyl sulfoxide
  • pY816 formulation (final concentration: 20 mg/kg): 100 mg pY816 or control peptide (customized by GenScript) was added to 12.5 ml phosphate buffered saline (cellgro, 21-040-CVCa), shaken until no precipitation, stored at -20°C, and used within one week. 0.1 ml was injected for every 20 g of mouse body weight.
  • mice tail lysis buffer 200 ⁇ l of mouse tail lysis buffer and 2 ⁇ l of Proteinase K (20mg/mL) to about 2mm of mouse tissue (toe or tail), mix thoroughly, and place in a 56°C oven for digestion for 6 hours or overnight. Then, place in a metal bath at 95°C to inactivate proteinase K and store at 4°C.
  • 2X Taq PCR Master Mix kit (Tiangen, KT211): 7 ⁇ l 2X Taq PCR Master Mix; 0.5 ⁇ l each of F-end and R-end primers; 5 ⁇ l ddH2O; 1 ⁇ l template; total volume 14 ⁇ l.
  • Primer information is as follows:
  • Emx1-F GCAAGAACCTGATGGACATGTTCAG
  • Emx1-R GCAATTTCGGCTATACGTAACAGGG
  • TrkB-F ATGTCGCCCTGGCTGAAGTG
  • TrkB-R ACTGACATCCGTAAGCCAGT
  • Cdkl5 fl/Y ;Emx1-Cre and Cdkl5 fl/Y ;CaMK2 ⁇ -iCre mice were monitored by video from adulthood, and Cdkl5 fl/Y ;CaMK2 ⁇ -CreER mice were monitored by video from the time of tamoxifen injection.
  • the observations were made under single-blind conditions and the observed seizure numbers, times, and seizure levels were recorded. The monitoring was performed for 1 hour every day (5 days a week). After tamoxifen injection, Cdkl5 fl/Y ;TrkB ;Cam2 ⁇ -CreER mice and their control groups were video recorded for 24 hours every week.
  • mice were graded according to the Racine standard: Grade 1, with shaking of the whiskers and face; Grade 2, with obvious nodding movements; Grade 3, unilateral forelimb spasm, with the tail erect; Grade 4, forelimb spasm, obvious myotonia; Grade 5, loss of limb control, forced clonus, and convulsions. Because first- and second-degree seizures are mild and can be easily missed during video recording, only third-degree or higher seizures were considered spontaneous seizures in our study (Racine, 1972).
  • the mouse head was fixed to a stereotaxic apparatus, the skull was exposed, two small holes were polished at the electrode implantation point, the EEG electrode was screwed into the small hole, and fixed to the skull with dental cement.
  • the EMG electrode was inserted into the trapezius muscle with forceps. Three days after the surgery, the adaptation was recorded for two consecutive days. EEG and EMG were recorded for freely moving mice, and the data were collected at a frequency of 1 kHz using Spike2 software (CED Ltd., Micro1401mkII).
  • Protein content was determined using the BCA protein quantification kit (Tiangen, PA115). Prepare BCA working solution, mix reagents A and B at a volume ratio of 50:1; dilute the BSA standard with a solution of the same buffer system as the sample, and dilute the sample 5-10 times; use a 96-well plate, with each well containing 25 ⁇ l of diluted sample or BSA standard and 200 ⁇ l of BCA working solution, mix thoroughly and cover Place in a 37°C oven for 30 minutes; after 30 minutes, use an ELISA reader (Molecular Devices) to detect the absorbance at 562 nm; calculate the protein concentration of the sample based on the standard curve.
  • BCA protein quantification kit Triangen, PA115.
  • the sample concentration was adjusted to level using protein loading buffer and heated at 80°C for 7 minutes.
  • An electrophoresis device equipped with SDS-PAGE gel plates (VE-180 vertical electrophoresis tank, Shanghai Tianneng Technology Co., Ltd., China) was prepared in advance and 1X electrophoresis buffer was added.
  • the cooked protein samples were then added to the lanes of the gel plates in sequence using a pipette.
  • the parameters for electrophoresis were: constant voltage 80V for 30 minutes in the first stage; constant voltage 110V for 100 minutes in the second stage.
  • PageRuler TM Plus Prestained Protein Ladder (Thermo Scientific, 26619) was used as the electrophoresis reference standard.
  • the protein was transferred to the PVDF membrane using a constant current mode of 350mA for 3 hours.
  • skim milk powder Yili
  • BSA aladdin, B265991
  • a 5% blocking solution with TBST buffer was used to prepare a 5% blocking solution with TBST buffer, and the solution was blocked at room temperature for 1 hour.
  • the corresponding protein primary antibody was diluted in the blocking solution and incubated overnight at 4°C.
  • the membrane was washed three times at room temperature using TBST buffer on a bed for 5-7 minutes each time.
  • the corresponding protein secondary antibody was dissolved in blocking solution and incubated at room temperature for 1-2 hours. After incubation, the membrane was washed three times and exposed using Pro-Light HRP chemiluminescent detection reagent (Tiangen, PA112) in a chemiluminescent imager (Analytik Jena AG).
  • RNA Master SYBR Green I kit (Roche, 03064760001) was used for RT-PCR reaction.
  • the reaction system was 10 ⁇ L SYBR Green Mix, 1 ⁇ l primer, 2 ⁇ l cDNA template, 6 ⁇ l ddH2O, and the total volume was 20 ⁇ l.
  • the reaction procedure was 95°C, 5 min; 95°C, 10 s, 60°C, 20 s, 72°C, 20 s, and the second to fourth steps were cycled 40 times; 95°C, 5 s; 65°C, 1 min.
  • Primer information is as follows:
  • GAPDH-R TTGGGGGTAGGAACACGGAAGG
  • Golgi staining was performed using the FD Rapid Golgi Stain TM Kit (FD Neuro Technologies, PK401). After anesthetizing the mouse, open the chest cavity and expose the heart. Use a syringe to inject the PBS solution from the left ventricle and out of the right atrium until the lungs and liver turn white. Cut off the head with scissors to expose the skull, and use the tip of the scissors to gently slide along the exposed inner surface of the bone from the cerebellum to the olfactory bulb, peel off the brain, and quickly remove the complete brain tissue.
  • FD Rapid Golgi Stain TM Kit FD Neuro Technologies, PK401
  • the slicing parameters were as follows: speed, 0.5-0.7 mm/s; amplitude, 1 mm; brain slice thickness, 150 ⁇ m; continuous collection.
  • the brain slices were attached to gelatin-coated slides and dried in the dark for more than 3 days before staining. First, the slides were placed in double distilled water twice, each for 4 minutes; then the slides were placed in a staining mixture for 10 minutes.
  • the staining mixture contained Solution D, Solution E and double distilled water in a ratio of 1:1:1; the slides were placed in double distilled water for two 4-minute washes before entering the dehydration process.
  • mice were anesthetized, the chest cavity was opened to expose the heart.
  • the perfusion needle infused the perfusion solution from the left ventricle and out of the right atrium.
  • PBS was perfused until the lungs and liver turned white, and then 4% paraformaldehyde (Acmec, P35120) was changed to continue the perfusion.
  • the mice were visibly in a rigid state. Cut off the head with scissors, cut the skin to expose the skull, and use the tip of the scissors to gently slide along the exposed inner surface of the bone from the cerebellum to the olfactory bulb to peel off the brain.
  • the brain was taken out and soaked in 10ml 4% paraformaldehyde fixative for 24h, and placed in a gradient dehydration of 10%, 20%, and 30% sucrose solution. Cool the slicer to a suitable temperature, flatten the bottom of the brain, absorb excess liquid with filter paper, place it in an embedding tank, add an appropriate amount of OCT embedding agent (Servicebio, G6059) to submerge the tissue, place it on a quick freezing table, and freeze it for 30 minutes. After removal, apply embedding agent to the slice base, fix the brain tissue, and place it in a quick freezer for 30 minutes. Place the frozen brain tissue on the slicer holder and flatten the tissue. Adjust the anti-roll plate to the appropriate position and start slicing. Set the thickness of the brain slice to 40 ⁇ m and collect the brain slice in PBS solution. Wash it twice with PBS, 10 minutes each time, attach the brain slice to a gelatin-coated slide, and dry it.
  • OCT embedding agent Servicebio, G6059
  • Dendritic spine density was calculated by dividing the total number of dendritic spines by the length of the dendritic shaft. The proportion of each dendritic spine subtype was calculated as the percentage of the total dendritic spines on the dendritic segment.
  • mice were anesthetized with isoflurane (Sigma, 1349003). After opening the chest, a small incision was made in the right atrium, and perfusion was performed with a syringe from the left ventricle near the apex of the heart. The perfusion fluid used was pre-cooled oxygenated (95% O2 + 5% CO2) slicing fluid until the lungs and liver turned white. The brain tissue was quickly peeled off and placed in pre-oxygenated pre-cooled slicing fluid for cooling. The brain tissue was cut into 300 ⁇ m coronal slices using a vibrating slicer (Leica, VT1200S) with continuous oxygenated pre-cooled slicing fluid, mainly retaining the hippocampus.
  • a vibrating slicer Leica, VT1200S
  • the cut brain slices were transferred to 32°C continuously oxygenated slicing fluid for incubation for 12 minutes. At this time, the brain slices can be divided into two halves along the sagittal plane. Then they were transferred to 25°C repair HEPES artificial cerebrospinal fluid for incubation for at least one hour. Electrophysiological recordings can be performed after one hour. The brain slices can be maintained for 7-8 hours and must always be in oxygen-saturated repair HEPES artificial cerebrospinal fluid. During the recording process, the brain slices were perfused with oxygen-saturated artificial cerebrospinal fluid at a rate greater than or equal to 2 ml/min.
  • Example 1 Spontaneous epilepsy phenotype can be observed in mice with CDKL5 knockout in forebrain excitatory neurons
  • CDKL5 deficiency In order to simulate the symptoms of patients with CDKL5 deficiency and conduct basic research on CDKL5 deficiency, researchers have constructed a variety of CDD mouse models since the discovery of the CDKL5 gene, mainly including Cdkl5 knockout mice, point mutation mice that simulate patients, and conditional knockout mice. These mouse models have proven that CDKL5 is essential in neurological functions such as movement, learning and memory, and social interaction, but the core clinical symptom of CDD patients, spontaneous epilepsy, has not been reproduced.
  • the conditional knockout mouse model (Conditional knockout, referred to as cKO), can knock out the target gene at a specific time or tissue-specifically to meet the specific needs of researchers and conduct more targeted research.
  • Emx1-ires-Cre and CaMK2 ⁇ -iCre tool mice have flox sequences inserted in the same direction on both sides of exon 6 of Cdkl5;
  • Emx1-cre tool mice express Cre recombinase in excitatory neurons in the cortex and hippocampus, and glial cells in the globus pallidus from embryonic stage E10.5;
  • CaMK2 ⁇ -iCre tool mice express Cre recombinase in glutamatergic excitatory neurons in the forebrain after birth.
  • mice of the Cdkl5 fl/Y ;Emx1-Cre strain will have spontaneous grand mal seizures; at 3 months of age, about 50% of the mice can be observed to have epileptic seizures; while some mice of the Cdkl5 fl/Y ;CaMK2 ⁇ -iCre mice have spontaneous grand mal seizures starting from 7 weeks of age, and about 50% of the mice can be observed to have epileptic seizures at 3 months of age.
  • this spontaneous epilepsy starts with mild to moderate clonus and forelimb tremor in the early stage, and gradually develops into a high-grade generalized seizure (Figure 4C, Figure 5C).
  • mice were overly sensitive to vibration, touch, and sound, which is very close to the irritable clinical signs of CDD patients.
  • EEG electroencephalogram
  • EMG electromyography
  • mice Both strains of mice can be observed to have sudden death caused by severe spontaneous epileptic seizures, which may occur in the early life of CDD model mice (2-3 months old). Some mice also have physical dysfunction due to frequent spontaneous epilepsy, and are unable to move and eat until they die. At 6-7 months of age, approximately 70% of Cdkl5 fl/Y ;Emx1-Cre and Cdkl5 fl/Y ;CaMK2 ⁇ -iCre mice died ( FIG. 4D , FIG. 5D ).
  • Example 2 Excitation-inhibition imbalance in the hippocampal dentate gyrus of forebrain excitatory neurons in Cdk15 knockout mice
  • the dentate gyrus receives its main input from the entorhinal cortex through the perforant path with glutamate synapses, and its molecular layer granule cell proximal dendrites form synapses with perforant path axons. Subsequently, granule cells project to the CA3 region through mossy fibers, which terminate on pyramidal neurons of CA3. Under physiological conditions, there are few direct interconnections between granule cells. In addition, granule cells themselves have a high resting membrane potential and strong GABA receptor-mediated inhibition, which makes them considered to be shock absorbers between the entorhinal cortex and CA3.
  • the hippocampal circuit presents an excitatory/inhibitory imbalance phenotype, which has appeared in many epilepsy models and also exists in other Cdkl5 knockout models. This suggests that forebrain excitatory neurons, as a key cell type for CDD-related epilepsy, will enhance excitatory synaptic transmission in the dentate gyrus and disrupt the balance of excitatory/inhibitory synaptic transmission in the hippocampal circuit, which may be the circuit basis for the spontaneous epileptic seizures in these two Cdkl5 conditional knockout mouse strains.
  • Example 3 Spontaneous epilepsy phenotype can be observed in tamoxifen-induced Cdkl5 fl/Y ; CaMK2 ⁇ -CreER mice
  • CDKL5 not only plays an important role in the development of the nervous system, but also maintains a high expression level in adulthood. Studies have shown that restoring knocked-out Cdkl5 in adulthood can greatly improve the impaired cognitive function of mouse models, indicating that CDKL5 also plays an important role in neural maturation. But is CDKL5 protein in neural maturation also critical for its spontaneous epilepsy phenotype?
  • Cdkl5 fl/Y ;Emx1-Cre and Cdkl5 fl/Y ;CaMK2 ⁇ -iCre mice the main causes of death in Cdkl5 fl/Y ;CaMK2 ⁇ CreER mice include sudden death from epilepsy and physical dysfunction.
  • the control group i.e., the Cdkl5 fl/Y mice injected with tamoxifen and the Cdkl5 fl/Y ;CaMK2 ⁇ -CreER mice injected with corn oil, no abnormal behavior, synchronized discharges or abnormal deaths were observed.
  • CDD spontaneous epilepsy associated with CDD is closely related to the functional defect of CDKL5 during neural maturation.
  • CDD has been considered a simple neurodevelopmental disease, but our results prove that CDKL5 protein is critical to circuit excitability during neural maturation.
  • the lack of CDKL5 protein during neural maturation can also induce circuit hyperexcitability and lead to epilepsy. Therefore, the study of CDD-related epilepsy should not only consider its neurodevelopmental stage, but also the dysfunction during its neuromaturation stage.
  • the tamoxifen-induced adult Cdkl5 knockout mouse model has the advantages of short onset latency (two weeks of action) and high spontaneous epileptic seizure rate (100% seizure rate), so it can well simulate the patient's seizure pattern and help study the mechanism of CDD-related epilepsy.
  • forebrain knockout of Cdkl5 will enhance the excitatory transmission of the hippocampal circuit, but it is still unknown whether knockout during neural maturation still has similar hyperexcitatory reactions.
  • Cdkl5 fl/Y CaMK2 ⁇ -CreER mice as the subject and performed whole-cell recordings on them one week after tamoxifen injection. At this time, CDKL5 in the forebrain tissue has been knocked out, but spontaneous epilepsy has not been observed.
  • Example 5 The morphology of dentate gyrus granule cells in tamoxifen-induced Cdk15 fl/Y ; CaMK2 ⁇ -CreER mice is normal
  • dendritic spines are closely related to the excitability of the circuit. Their morphology is affected by many factors and is in a constant adjustment process.
  • the morphological abnormalities of neuronal dendritic spines are an important part of epilepsy research. In different animal models, the morphological abnormalities of dendritic spines in multiple brain regions of the forebrain can be observed, including the cerebral cortex, the CA1 region of the hippocampus, and the dentate gyrus.
  • Dendritic spines are the transmission sites of excitatory synapses, which can receive information and form synaptic connections, and are considered to be the basis of synaptic plasticity.
  • Dendritic spines include three morphologies: thin, mushroom, and stubby. The thin type is considered to be an immature state, while the stubby and mushroom types are mature types.
  • Dendritic spine density analysis is widely used in neuroscience research and is an important means to evaluate the functional plasticity of synapses in the central nervous system. Through the Golgi staining experiment, using the silver-loving properties of nerve cells, we can The morphology of neurons and the various types of dendritic spines are well documented. Although the number of dendritic spines is not directly equivalent to the amount of synaptic activity, the analysis of dendritic spine density and type can be used to assess changes in synaptic connection strength and synaptic plasticity.
  • Example 6 Abnormal activation of the hippocampal BDNF-TrkB signaling pathway is the key to enhancing excitatory synaptic transmission in the dentate gyrus of the hippocampus
  • BDNF Brain-derived neurotrophic factor
  • TrkB Brain-derived neurotrophic factor
  • BDNF affects synaptic transmission and changes the morphological characteristics of neurons, leading to excessive excitation of the hippocampal circuit, and this abnormal excitation in turn promotes the increase in BDNF expression levels, forming a pathological positive feedback.
  • Trk receptor inhibitor K252 was used to verify the blocking Can the BDNF-TrkB signaling pathway rescue the enhanced excitatory synaptic transmission of the circuit caused by tamoxifen-induced Cdkl5 knockout?
  • sEPSC spontaneous excitatory postsynaptic current
  • mEPSC miniature excitatory postsynaptic current
  • Trk receptor inhibitor K252a can well rescue the increase in sEPSC/mEPSC frequency caused by Cdkl5 knockout without affecting the excitatory synaptic transmission of the control group, and improve the enhanced excitatory synaptic transmission of dentate gyrus granule cells.
  • K252a is a broad-spectrum Trk inhibitor that may interfere with the function of other neurotrophic factors and affect subsequent experimental designs
  • ANA-12 a specific antagonist of TrkB.
  • sEPSCs spontaneous excitatory postsynaptic currents
  • mEPSCs miniature excitatory postsynaptic currents
  • Example 7 Inhibition of the BDNF-TrkB signaling pathway reduces epileptic activity in Cdkl5fl/Y;CaMK2 ⁇ -CreER mice
  • TrkB receptor antagonist ANA12 intervenes in the abnormal activation of the BDNF-TrkB signaling pathway by inhibiting the TrkB receptor.
  • the frequency of epileptic activity in the Cdkl5 fl/Y ;CaMK2 ⁇ -CreER tamoxifen-injected mice was significantly reduced compared with 12 hours before injection, and its anti-epileptic effect was comparable to that of the classic anti-epileptic drug valproic acid (Figure 16A-C), indicating that inhibiting TrkB receptors after spontaneous epilepsy can also effectively reduce CDD-related epileptic activity, suggesting that the BDNF-TrkB signaling pathway can be an excellent target for the treatment of CDD-related epilepsy.

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Abstract

La présente invention se rapporte à l'utilisation d'une dans la prévention et/ou le traitement de l'épilepsie liée à un trouble lié à une déficience en CDKL5. Plus particulièrement, la présente invention concerne l'utilisation d'un inhibiteur de la voie de signalisation BDNF-TrkB dans la préparation d'une composition ou d'une préparation. La composition ou la préparation est utilisée pour prévenir et/ou traiter l'épilepsie liée à un trouble lié à une déficience en CDKL5. La présente invention découvre pour la première fois que l'inhibiteur de la voie de signalisation de BDNF-TrkB peut prévenir et/ou traiter l'épilepsie liée à un trouble de déficience en CDKL5.
PCT/CN2024/097792 2023-06-20 2024-06-06 Utilisation de voie de signalisation bdnf-trkb dans la prévention et/ou le traitement de l'épilepsie liée à un trouble de déficience en cdkl5 Pending WO2024260261A1 (fr)

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US10525024B2 (en) * 2014-08-15 2020-01-07 The Johns Hopkins University Methods for rescuing phenobarbital-resistance of seizures by ANA-12 or ANA-12 in combination with CLP290

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US10525024B2 (en) * 2014-08-15 2020-01-07 The Johns Hopkins University Methods for rescuing phenobarbital-resistance of seizures by ANA-12 or ANA-12 in combination with CLP290

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* Cited by examiner, † Cited by third party
Title
LIU, HUI ET AL.: "The Brain Derived Neurotrophic Factor and Epilepsy", LIN CHUANG YU BING LI ZA ZHI = FOREIGN MEDICAL SCIENCES (PATHOPHYSIOLOGY AND CLINICAL MEDICINE), CN, vol. 22, no. 6, 31 December 2002 (2002-12-31), CN , pages 618 - 620, XP009559945, ISSN: 1001-1773 *
MIAO, LU: "k252a Inhibits BDNF-TrkB Signaling Pathway to Effect the Expression of Arc in Electrical Stimulation Kindled Rats and Epilepsy", WANFANG MASTER'S THESES, 28 November 2012 (2012-11-28), XP093251821 *

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