Use of nav1.1 agonists in the manufacture of a medicament for treating autism

Abstract

The application discloses application of Nav1.1 agonists in preparation of medicines for treating autism, wherein the Nav1.1 agonists are Compound 4 and / or Lu AE98314. Lu AE98314 and Compound 4 can show obvious curative effects on treatment of autism through different modes of administration due to their special structures. The application has great significance for treatment of autism patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to the application of Compound 4 and Lu AE98314, two Nav1.1 agonists, in the preparation of a medicament for treating autism. BACKGROUND

[0002] Autism is a pervasive developmental disorder characterized by social interaction disorders, communication disorders, narrow interests and stereotyped behavior patterns. Autism is a type of autism spectrum disorder (ASD), and the current cause of the disease is not very clear, which may be related to genetic variation and environmental factors. A small number of ASD cases are associated with mutations in a single gene (Geschwind, D.H., and Levitt, P. (2007). Autism spectrum disorders: developmental disconnection syndromes. Curr Opin Neurobiol 17, 103-111). ASD has no effective treatment method so far, and the effect of clinical behavior training and rehabilitation method is uncertain. Therefore, in-depth study of the pathogenesis of autism model mice and development of drugs targeting key functional changes are of great significance for the treatment of autism patients.

[0003] Point mutations in the postsynaptic cell adhesion molecule 3 (neuroligin 3, NL3) (R451C site mutation) (Jamain, S., Quach, H., Betancur, C., Rastam, M., Colineaux, C., Gillberg, I.C. (2003). Mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 are associated with autism. Nat Genet, 34(1), 27-29.) and partial deletions (Levy, D., Ronemus, M., Yamrom, B., Lee, Y.H., Leotta, A., Kendall, J., Wigler, M. (2011). Rare de novo and transmitted copy-number variation in autistic spectrum disorders. Neuron, 70(5), 886-897.) have been strongly associated with ASDs.NL3 R451C site knock-in (KI) mice exhibit hyperactivity, repetitive stereotypy and social novelty-seeking behavior disorders (Tabuchi, K., J. Blundell, M. R. Etherton, R. E. Hammer, X. Liu, C. M. Powell and T. C. Sudhof (2007). "A neuroligin-3 mutation implicated in autism increases inhibitory synaptic transmission in mice." Science 318(5847): 71-76.; Etherton, M., C. Foldy, M. Sharma, K. Tabuchi, X. Liu, M. Shamloo, R. C. Malenka and T. C. Sudhof (2011). "Autism-linked neuroligin-3 R451C mutation differentially alters hippocampal and cortical synaptic function." Proc Natl Acad Sci U S A 108(33): 13764-13769.; Rothwell, P. E., M. V. Fuccillo, S. Maxeiner, S. J. Hayton, O. Gokce, B. K. Lim, S. C. Fowler, R. C. Malenka and T. C. Sudhof (2014). "Autism-associated neuroligin-3 mutations commonly impair striatal circuits to boost repetitive behaviors." Cell 158(1): 198-212.).

[0004] Parvalbumin (PV) positive interneurons are strong regulators of local network activity and are key factors in the generation of gamma oscillations (30-80 Hz), and dysfunction of which can lead to impaired gamma oscillations, affecting cognitive function, which is closely related to various neuropsychiatric diseases, such as ASD (Buzsaki, G., and Wang, X. J. (2012). Mechanisms of gamma oscillations. Annu Rev Neurosci 35, 203-225).

[0005] Nav1.1 is a voltage-gated sodium channel subunit, primarily expressed in the central nervous system (Catterall, WA (2017). Forty Years of Sodium Channels: Structure, Function, Pharmacology, and Epilepsy. Neurochem Res 42, 2495-2504). It is highly expressed in PV-positive fast-spiking (FS) interneurons in the brain, controlling membrane depolarization and action potential firing (Ogiwara, I., Miyamoto, H., Morita, N., Atapour, N., Mazaki, E., Inoue, I., Takeuchi, T., Itohara, S., Yanagaawa, Y., Obata, K., et al. (2007). Nav1.1 localizes to axons of parvalbumin-positive inhibitory interneurons: a circuit basis for epileptic seizures in mice carrying an Scn1agene mutation. J Neurosci 27, 5903-5914). A relatively common cause of Nav1.1 mutations in the SCN1A gene is epilepsy, such as generalized tonic-clonic seizures with febrile seizures (GEFS+).

[0006] There are currently no approved Nav1.1 agonists on the market or in clinical trials. This may be due to the lack of homomorphic selectivity in treatments targeting the Nav channel, leading to side effects beyond the target. Summary of the Invention

[0007] This invention delves into the pathogenesis of autism in mouse models, develops drugs targeting key functional alterations, and provides the application of Compound 4 and Lu AE98314, two Nav1.1 agonists, in the preparation of drugs for treating autism, which is of great significance for the treatment of autism patients.

[0008] This invention reveals that not all Nav1.1 agonists are effective in treating autism; however, the compounds Compound 4 and Lu AE98314, which were found to be effective in this study, happen to be Nav1.1 agonists. Compound 4, compared to Lu AE98314, exhibits stronger selectivity, a lower effective concentration, and the ability to cross the blood-brain barrier. When mice were intraperitoneally injected with 30 mg / kg for one hour, a three-component social behavior test was performed. The results showed that Compound 4 significantly improved social impairment in KI mice. We further explored the possible mechanism by which Compound 4 rescues social behavior, finding that in acute ex vivo brain slices, 0.3 μM Compound 4 significantly increased the excitability of PV-positive FS interneurons in the mPFC of KI mice to the WT level. This suggests that Compound 4 may rescue the social novelty deficit in autism model mice by increasing the excitability of PV-positive interneurons, which has significant implications for the treatment of autism patients.

[0009] The use of Nav1.1 agonists in the preparation of drugs for treating autism, wherein the Nav1.1 agonists are Compound4 and / or Lu AE98314;

[0010] The structure of Compound 4 is shown in the following formula:

[0011]

[0012] The structure of the Lu AE98314 is shown in the following formula:

[0013]

[0014] As a general inventive concept, the present invention also provides a medicament suitable for treating autism, the medicament containing components Compound 4 and / or Lu AE98314. In this medicament, Compound 4 and Lu AE98314 serve as active components for treating autism.

[0015] The drug for treating autism described in this invention can be a solid or liquid preparation. It can be prepared according to conventional pharmaceutical production methods to form the required dosage form. Pharmaceutically acceptable carriers can be added during the preparation process. The form can be diverse, such as tablets, capsules, oral liquids, injections, infusions, ointments, lyophilized powder injections, liniments, or suppositories.

[0016] The medication for treating autism is either a gastrointestinal or non-gastrointestinal formulation, and can be administered in unit doses. For external use, non-gastrointestinal administration methods include injection, such as intravenous injection, intraperitoneal injection, intramuscular injection, acupoint injection, and subcutaneous injection.

[0017] The medication for treating autism may also include pharmaceutically acceptable carriers and / or excipients. Pharmaceutically acceptable carriers refer to conventional pharmaceutical carriers, such as diluents; fillers such as sucrose and starch; binders such as hydroxypropyl cellulose and starch paste; humectants such as magnesium stearate and micronized silica; absorption enhancers such as polysorbate and lecithin; and surfactants such as sorbitan and beroxam. Other excipients, such as sweeteners and flavorings, may also be added to the medication for treating autism.

[0018] Compared with existing technologies, the main advantages of this invention include: This invention has found that Lu AE98314 and Compound 4, due to their unique structures, can exhibit significant therapeutic effects on autism when administered via different routes. While AA43279 is also a Nav1.1 agonist and can cross the blood-brain barrier, its therapeutic effect is significantly poor, and it lacks application prospects. Surprisingly, this invention has found that Compound 4 has stronger selectivity, a lower effective concentration, and can cross the blood-brain barrier, demonstrating significant efficacy in the treatment of autism. This invention is of great significance for the treatment of autistic patients. Attached Figure Description

[0019] Figure 1 (a) Figure 1 (d) shows the structure of AA43279 and its response to sodium channels with different voltage gates, respectively; Figure 1 (b) Figure 1 (e) shows the structure of Lu AE98314 and its response to sodium channels with different voltage gates, respectively; Figure 1 (c) Figure 1 (f) shows the structure of Compound 4 and its response to sodium channels with different voltage gates, respectively;

[0020] Figure 2The diagram illustrates the different effects of AA43279, Lu AE98314, and Compound 4 on social novelty deficits in adult KI mice. (a) shows a schematic diagram of the brain region where the surgically implanted drug delivery cannula was placed, and a flowchart of the social rescue effect assay for AA43279 via cannula administration. (bc) shows that AA43279 can slightly improve social novelty deficits in KI mice. (b) shows the sociality difference index, and (c) shows the social novelty difference index. (d) shows a schematic diagram of the brain region where the surgically implanted drug delivery cannula was placed, and a flowchart of the social rescue effect assay for Lu AE98314 via cannula administration. (ef) shows that Lu AE98314 can rescue social novelty deficits in KI mice. (e) shows the sociality difference index, and (f) shows the social novelty difference index. (g) shows a flowchart of the rescue effect assay using a three-box social assessment one hour after intraperitoneal injection of Compound 4. (hi) shows that Compound 4 can rescue social novelty deficits in KI mice. (h) shows the sociality difference index, and (i) shows the social novelty difference index. The values ​​in the bars represent the number of mice. *P<0.05, **P<0.01. Statistical method: one-way ANOVA. Data representation: mean ± sem.

[0021] Figure 3The effects of AA43279, Lu AE98314, and Compound 4 on the excitability of PV-positive FS interneurons in adult KI mice are shown. (ab) Brain slices were pre-incubated with 30 μM AA43279 for 30 minutes, and the excitability of FS interneurons in adult mice was recorded. (a) Figure shows the dotted line graph of action potential firing recorded on WT mice (left) and the histogram of the total number of action potentials generated by depolarized square wave stimulation (right). (b) Figure shows the dotted line graph of action potential firing recorded on KI mice (left) and the histogram of the total number of action potentials generated by depolarized square wave stimulation (right). (cd) Brain slices were pre-incubated with 30 μM Lu Thirty minutes after AE98314, the excitability of interneurons in the FS region of adult mice was recorded. (c) Figure shows the dotted line graph of action potential firing recorded on WT mice (left) and the histogram of the total number of action potentials generated by depolarized square wave stimulation (right). (d) Figure shows the dotted line graph of action potential firing recorded on KI mice (left) and the histogram of the total number of action potentials generated by depolarized square wave stimulation (right). *P<0.05, **P<0.01, statistical method: two-tailed t-test; (e) Thirty minutes after pre-incubation of brain slices with 0.3μM Compound 4, the dotted line graph of action potential firing recorded on adult mice (left) and the histogram of the total number of action potentials generated by depolarized square wave stimulation (right) were recorded. The four sets of data were each from three pairs of littermates. *P<0.05, statistical method: one-way ANOVA. Data representation: mean ± sem. Detailed Implementation

[0022] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Operating methods not specifically specified in the following embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0023] This invention selected three Nav1.1 agonists for experiments. AA43279 has the chemical formula C 12 H 12 N₂O₂S, CAS number 354812-16-1, molecular weight 248.30, can cross the blood-brain barrier. Lu AE98314 has the chemical formula C₂O₂. 21 H 23 N5O3S, CAS number 849000-18-6, molecular weight 425.50, cannot cross the blood-brain barrier. Compound 4 has the chemical formula C. 24 H 23F3N4O, CAS number 2332897-85-3, with a molecular weight of 440.46, can cross the blood-brain barrier. The structures of AA43279, Lu AE98314, and Compound 4, and their responses to sodium channels with different voltage gating methods, are as follows: Figure 1 As shown. AA43279 showed a high response to Nav1.1, as well as Nav1.5 and Nav1.6, and some response to Nav1.2. Lu AE98314 showed a high response to Nav1.1 at lower concentrations, but also a high response to Nav1.5, and some response to Nav1.2. Compound 4 showed a high response to Nav1.1 at even lower concentrations than Lu AE98314, and some response to Nav1.3, exhibiting higher specificity. This invention investigated the effects of AA43279 and Lu AE98314 on social behavior in autistic model mice via intraperitoneal injection (30 mg / kg). The results showed that intraperitoneal injection of AA43279 and Lu AE98314 did not improve the social novelty deficit in KI mice. This may be because Lu AE98314 cannot cross the blood-brain barrier, and while AA43279 can cross the blood-brain barrier, it is ineffective at a low concentration of 30 mg / kg. Based on this, we used surgical implantation of a drug delivery cannula to explore the effects of AA43279 and Lu AE98314 on social behavior in autistic model mice. Figure 2 af). One week after surgery, the three-way social test was performed. In the control group, KI mice showed normal social ability compared to WT mice half an hour after local brain injection of the control solvent, but exhibited abnormal social novelty, i.e., they could not distinguish well between familiar and unfamiliar mice, thus retaining the defective phenotype and ruling out the possibility that surgery might have affected social behavior. Figure 2 bc and ef). Half an hour after administration of AA43279, socialization and social novelty in WT mice were not affected, but social novelty deficits in KI mice were improved, though not significantly. Figure 2 bc). Half an hour after administration of Lu AE98314, socialization and social novelty in WT mice were not affected, but social novelty deficits in KI mice were significantly improved. Figure 2 However, considering its inability to cross the blood-brain barrier, which increases the difficulty of delivery and greatly limits its drug-likeness, this invention uses the same intraperitoneal injection (30 mg / kg) method to explore the effect of Compound 4 on the social behavior of autistic model mice. Figure 2 In the control group of the three-box social experiment, KI mice showed normal social abilities compared to WT mice, but exhibited abnormal social novelty. Figure 2Hi). One hour after Compound 4 administration, socialization and social novelty did not affect WT mice, but it significantly improved social novelty deficits in KI mice. Figure 2 (hi). It is evident that Compound4's drug delivery method is more convenient and has greater application prospects.

[0024] This invention also further explores the possible mechanisms by which Nav1.1 agonists rescue social behavior.

[0025] To label PV-positive interneurons, we used the PV-cre / Ai14 mouse strain; therefore, all fluorescently labeled neurons were PV-positive FS interneurons. Fluorescently labeled mice were generated by crossing NL3 R451CKI mice with PV-cre / Ai14 mice, and all electrophysiological experiments were conducted using these mice.

[0026] Acute ex vivo brain slices with a thickness of 300 μm were prepared for electrophysiological recording. We injected incremental depolarizing currents into FS interneurons and compared the number of action potentials fired by FS interneurons in WT and KI mice under the same depolarization conditions. FS interneurons were first current-clamped to maintain Vh at -70 mV, then stimulated with depolarizing square wave currents ranging from 150 pA to 600 pA, each wave lasting 500 ms, with increments of 50 pA each time. Recording began after incubation of the brain slices with 30 μM AA43279. Results showed that AA43279 had no significant effect on the excitability of PV-positive FS interneurons in both WT and KI mice. Figure 3 ab). Brain slices were incubated with 30 μM Lu AE98314 before recording began. The results showed that Lu AE98314 had no effect on the excitability of PV-positive FS interneurons in WT mice, while the excitability of PV-positive FS interneurons was significantly increased in KI mice. Figure 3 cd). Electrophysiological recordings were performed using the same method to compare the effects of Compound 4. In the control group, the number of action potentials fired by FS interneurons was significantly reduced in adult KI mice compared to WT mice. Figure 3 e). Brain slices were incubated with 0.3 μM Compound 4 before recording began. Results showed that Compound 4 restored the excitability of PV-positive FS interneurons in KI mice to WT levels. Figure 3e) Furthermore, the concentration used was much lower than that of Lu AE98314. Therefore, although both Lu AE98314 and Compound 4 can restore the excitability of PV-positive FS interneurons in KI mice to WT levels, to achieve the same effect, the dosage of Lu AE98314 is far greater than that of Compound 4 (Lu AE98314 dosage is 100 times that of Compound 4). In contrast, although AA43279 showed a trend of improvement, its therapeutic effect was significantly worse than that of Lu AE98314 and Compound 4.

[0027] Furthermore, it should be understood that after reading the above description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The application of Compound 4 in the preparation of drugs for treating autism, characterized in that, The structure of Compound 4 is shown in the following formula:

2. The application according to claim 1, characterized in that, The drug is a solid or liquid preparation.

3. The application according to claim 1, characterized in that, The drug is either a gastrointestinal or non-gastrointestinal dosage form.

4. The application according to claim 1, characterized in that, The drug includes pharmaceutically acceptable carriers and / or excipients.

Images