CASK disorder treating agent and treatment method

Abstract

This CASK disorder treating agent contains, as an active ingredient, at least one kinase inhibitor selected from the group consisting of JNK inhibitors and p38 MAPK inhibitors.

Description

CASK abnormality treatment agent and treatment method

[0001] The present invention relates to a therapeutic agent and method for CASK abnormality, and more specifically to a treatment for CASK abnormality by suppressing cerebellar hypoplasia in CASK abnormality.

[0002] CASK deficiency is a condition caused by abnormalities (deletions, point mutations, etc.) in the CASK gene, which encodes calcium / calmodulin-dependent serine protein kinase (CASK), located on the X chromosome at Xp11.4.

[0003] Commonly reported findings of CASK abnormalities include mental retardation and microcephaly with pontine and cerebellar hypoplasia (MICPCH) syndrome and X-linked intellectual disability (XLID), but there are also patients who do not fall into these categories.

[0004] MICPCH is a neurodevelopmental disorder characterized by congenital hypoplasia of the cerebellum and pons. While the majority of patients with MICPCH syndrome are female, male CASK knockout (KO) mice are lethal shortly after birth, which is thought to be why males with a CASK gene deficiency miscarry and are not born. Meanwhile, point mutations in the CASK gene have also been found in male patients, who often have associated symptoms such as autism and intellectual disability.

[0005] Genes on the X chromosome are known to undergo X-chromosome inactivation in females. X-chromosome inactivation occurs in the inner cell mass during early developmental stages, during the blastula stage. One of the two X chromosomes is randomly selected and inactivated in each cell, and subsequent divisions from that cell inherit the same X-chromosome inactivation pattern. Therefore, when a female is heterozygous for the CASK gene, mature individuals will have a random mixture of cells with normal CASK expression and cells lacking CASK. This may also be involved in the pathogenesis of female patients with MICPCH syndrome. The inventors conducted research using female heterozygous CASK-deficient mice and reported that X-chromosome inactivation results in a mosaic mixture of CASK-expressing and CASK-deficient cells in the brains of these mice, which may affect the neuropathological mechanism of MICPCH syndrome (Non-Patent Document 1).

[0006] Patent Document 1 discloses that neurodegenerative diseases and disorders such as spinocerebellar ataxia type 1 (SCA1) can be treated or prevented by administering a JNK inhibitor such as SP600125.

[0007] Patent Document 2 discloses that JNK inhibitors such as SP600125 inhibit neuronal degeneration in spinal muscular atrophy (SMA) caused by SMN deficiency, and that the brain-specific isoform JNK3 is particularly involved. It has also been confirmed that JNK activation was induced in both SMN-deficient neurons and ZPR1-deficient neurons when cerebellar granule neurons isolated from mouse cerebellum were cultured.

[0008] Patent Document 3 discloses a pharmaceutical composition containing a JNK inhibitor having a length of less than 150 amino acids for treating diseases or disorders strongly associated with JNK signaling, and lists motor neuron disease and spinocerebellar ataxia / atrophy as diseases.

[0009] US2022 / 0362335A1WO2010 / 151638A1WO2015 / 197194A1

[0010] Mori et al., Molecular Psychiatry 2019

[0011] The above-mentioned non-patent document 1 clarifies the mechanism by which mosaic defects in CASK in the cerebral cortex affect synaptic projections. However, the molecular and cellular mechanisms of cerebellar hypoplasia, which is a characteristic of MICPCH syndrome, and the neural circuit mechanisms and treatment methods that cause autistic symptoms as a developmental disorder caused by point mutations in the CASK gene have not yet been discovered.

[0012] The above Patent Documents 1 to 3 disclose that JNK inhibitors are used to treat neurodegenerative diseases and disorders, but do not mention their relationship with CASK abnormalities.

[0013] An objective of the present invention is to provide a therapeutic agent and a therapeutic method for CASK abnormalities.

[0014] In a study of cerebellar hypoplasia using CASK gene-deficient mice as an animal model of this syndrome, the inventors found that when cerebellar granule cells from CASK-deficient mice were cultured, the granule cells underwent cell death over time. Hoping that suppressing this CASK-deficiency-induced cerebellar granule cell death might lead to a treatment for cerebellar hypoplasia in MICPCH syndrome, they added candidate drugs to the culture medium and tested their inhibitory effects on cell death. They found that a c-Jun N-terminal kinase (JNK) inhibitor and a p38 MAPK inhibitor suppressed cerebellar granule cell death, leading to the completion of the present invention.

[0015] Furthermore, when a JNK inhibitor or a p38 MAPK inhibitor was injected into the cerebellum of CASK-deficient mice, cerebellar granule cell death was suppressed in vivo. Currently, there is no treatment for cerebellar hypoplasia in CASK disorders, and this discovery is the first example in the world of suppressing cerebellar cell death caused by CASK deficiency.

[0016] To achieve the above-mentioned objects, the present invention encompasses, for example, the following aspects. Item 1. A therapeutic agent for CASK abnormality, comprising as an active ingredient at least one kinase inhibitor selected from the group consisting of a JNK inhibitor and a p38 MAPK inhibitor. Item 2. The therapeutic agent according to Item 1, wherein the CASK abnormality includes MICPCH syndrome. Item 3. The therapeutic agent according to Item 1 or 2, which is used to suppress cerebellar hypoplasia in MICPCH syndrome. Item 4. A pharmaceutical composition for treating CASK abnormality, comprising the therapeutic agent according to any one of Item 1 to 3 and a pharmaceutically acceptable carrier. Item 5. A method for treating cerebellar hypoplasia due to CASK abnormality, using at least one kinase inhibitor selected from the group consisting of a JNK inhibitor and a p38 MAPK inhibitor. Item 6. A method for treating cerebellar hypoplasia due to CASK abnormality according to Item 5, which comprises injecting into the cerebellum a pharmaceutical composition containing at least one kinase inhibitor selected from the group consisting of a JNK inhibitor and a p38 MAPK inhibitor. Item 7. Item 7. A method for suppressing cerebellar hypoplasia due to CASK abnormality, comprising administering the therapeutic agent for CASK abnormality according to any one of Items 1 to 3. Item 8. Use of at least one kinase inhibitor selected from the group consisting of a JNK inhibitor and a p38 MAPK inhibitor for the manufacture of a pharmaceutical composition for the treatment of CASK abnormality.

[0017] According to the present invention, a new therapeutic agent and method for CASK abnormality can be provided.

[0018] Figure 1A is a micrograph showing the phenotype of cerebellar granule cells in CASK-deficient mice. Figure 1B is a micrograph showing the phenotype of cerebellar granule cells in CASK-deficient mice. Figure 1C is a graph showing the phenotype of cerebellar granule cells in CASK-deficient mice. n = 3 per group, **P < 0.01, *P < 0.05. Figure 2 is a graph showing the inhibition of granule cell death when cerebellar granule cells in CASK-deficient mice were cultured with the JNK inhibitor SP600125. n = 3 per group, **P < 0.01, *P < 0.05. Figure 3 is a graph showing the inhibition of granule cell death when cerebellar granule cells in CASK-deficient mice were cultured with different concentrations of the JNK inhibitor SP600125. ****: P < 0.0001, ns: not significant. Figure 4 is a graph showing the inhibition of granule cell death when cerebellar granule cells in CASK-deficient mice were cultured with the JNK inhibitor JNK-IN-8. Figure 5 is a graph showing the inhibition of granule cell death when CASK-deficient mouse cerebellar granule cells were cultured with the JNK inhibitor JNK-IN-8 at different concentrations. ****: P<0.0001, *: P<0.05, ns: no significant difference. Figure 6 is a graph showing the inhibition of granule cell death when CASK-deficient mouse cerebellar granule cells were cultured with the JNK inhibitor Tanzisertib. Figure 7 is a graph showing the inhibition of granule cell death when CASK-deficient mouse cerebellar granule cells were cultured with the JNK inhibitor Tanzisertib at different concentrations. ****: P<0.0001, ns: no significant difference. Figure 8 is a graph showing the inhibition of granule cell death when CASK-deficient mouse cerebellar granule cells were cultured with the p38 MAPK inhibitor Doramapimod. Figure 9 is a graph showing the inhibition of granule cell death when cerebellar granule cells from CASK-deficient mice were cultured with various concentrations of the p38 MAPK inhibitor, Doramapimod. ****: P<0.0001, ns: no significant difference. Figure 10 is a graph comparing the inhibitory effects of the four JNK inhibitors shown in Figures 2 to 9 on granule cell death. The concentrations of each JNK inhibitor were those that had the greatest inhibitory effect on cerebellar granule cell death: SP600125: 10 μM, JNK-IN-8: 1 μM, Tranzisertib: 20 μM, Doramapimod: 1 μM.Figure 11 shows the suppression of granule cell death in wild-type (WT) mouse cerebellar granule cells and CASK-deficient mouse cerebellar granule cells cultured with different concentrations of the JNK inhibitor JNK-IN-7. WT lenti: wild-type (WT) mouse cerebellar granule cells; WT iCre: wild-type (WT) mouse cerebellar granule cells transfected with Cre recombinase; fCASK lenti: cultured cerebellar granule cells from CASK floxed mice; fCASK iCRE: cultured cerebellar granule cells from CASK floxed mice transfected with Cre recombinase. ****: P<0.0001; #: difference between treated CASK knockout group and DMSO (untreated) CASK knockout group; ####: P<0.0001; ns: not significant; ND: not detected. Figure 12 is a graph showing the inhibition of granule cell death in wild-type (WT) mouse cerebellar granule cells and CASK-deficient mouse cerebellar granule cells cultured with different concentrations of the JNK inhibitor DB07268. WT lenti: wild-type (WT) mouse cerebellar granule cells, WT iCre: wild-type (WT) mouse cerebellar granule cells transfected with Cre recombinase, fCASK lenti: cultured cerebellar granule cells generated from CASK floxed mice, fCASK iCRE: cultured cerebellar granule cells generated from CASK floxed mice transfected with Cre recombinase. ****: P<0.0001, #: difference between treated CASK knockout group and DMSO (untreated) CASK knockout group, ###: P<0.001, #: P<0.05, ns: not significant, ND: not detected. Figure 13 is a graph showing the recovery of cerebellar granule cell density upon local injection of the JNK inhibitor JNK-IN-8 into the cerebellum of CASK-deficient mice. Clasping (spasticity) scores in CASK gene-deficient model mice. A. Photographs showing the contracture state of mice in each group. B. Average limb clasping scores for each group. ****: P<0.0001. n=7 per group.

[0019] The present invention will be described in detail below.

[0020] As used herein, the singular forms (a, an, the) include both the singular and the plural unless otherwise expressly stated herein or clearly contradicted by context.

[0021] In this specification, the term "comprise" is a concept that encompasses "consist essentially of" and "consist only of."

[0022] Unless otherwise defined herein, scientific and technical terms used shall have the meanings commonly used in the art. Generally, the terms and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genes and proteins described herein are those well known and commonly used in the art.

[0023] 1. At least one kinase inhibitor selected from the group consisting of JNK inhibitors and p38 MAPK inhibitors. In the present invention, "JNK inhibitor" refers to a kinase inhibitor that can block or prevent the action, function, or phosphorylation of c-Jun N-terminal kinase (JNK). "Inhibitor" does not include irreversible substances that destroy JNK molecules and / or kinase activity. Furthermore, "JNK activity" refers to the kinase activity of c-Jun N-terminal kinase (JNK), and "JNK inhibitor" refers to a substance that inhibits JNK activity. JNK has 10 isoforms derived from three genes, specifically, JNK1 (MAPK8) has four isoforms, JNK2 (MAPK9) has four isoforms, and JNK3 (MAPK10) has two isoforms.

[0024] JNK inhibitors include SP600125 (anthra[1,9]pyrazol-6(2H)-one or 1,9-pyrazoloanthrone; CAS No. 129-56-6), JNK-IN-1 (CAS No. 2745108-35-2), JNK-IN-7 (CAS No. 1408064-71-0), JNK-IN-8 (CAS No. 1410880-22-6), SR-3306 (N-[4-[3-(6-methylpyridin-3-yl)-1,2,4-triazol-1-yl]phenyl]-4-(3-morpholin-4-ylphenyl)pyrimidin-2-amine), TAT-JNK III (AS601245 (CAS No. 345987-15-7), AS602801 (CAS No. 1410880-22-6), and SR-3306 (N-[4-[3-(6-methylpyridin-3-yl)-1,2,4-triazol-1-yl]phenyl]-4-(3-morpholin-4-ylphenyl)pyrimidin-2-amine). No. 848344-36-5), AEG3482 (CAS No. 63735-71-7), D-JNKi (CAS No. 1198367-70-2), Tanzisertib (CAS No. 899805-25-5), XG-102 (D-JNK-1) (CAS No. 1445179-97-4), CC-401 (CAS No. 395104-30-0), Doramapimod (CAS No. 285983-48-4), DB07268 (CAS No. 929007-72-7).

[0025] In the present invention, the term "p38 MAPK inhibitor" refers to a kinase inhibitor that can inhibit or prevent the action, function, or phosphorylation of p38 MAP kinase. Furthermore, "p38 MAPK activity" refers to the kinase activity of p38 MAP kinase, and a "p38 MAPK inhibitor" refers to a substance that inhibits the kinase activity of p38 MAP kinase. Four p38 MAP kinases have been identified in mammals: p38-α (MAPK14), p38-β (MAPK11), p38-γ (MAPK12 / ERK6), and p38-δ (MAPK13 / SAPK4).

[0026] p38 MAPK inhibitors include, but are not limited to, Adezmapimod (CAS No. 152121-47-6), SB202190 (CAS No. 152121-30-7), Metformin (CAS No. 657-24-9) and its hydrochloride salt (CAS No. 1115-70-4), and Doramapimod (CAS No. 285983-48-4).

[0027] Doramapimod is a compound that can be classified as both a JNK inhibitor and a p38 MAPK inhibitor.

[0028] At least one kinase inhibitor selected from the group consisting of JNK inhibitors and p38 MAPK inhibitors may be in the form of a solvate with an organic solvent and / or water. When forming a hydrate, the solvate may be coordinated with any number of water molecules. For example, a monohydrochloride monohydrate may be mentioned.

[0029] In the present invention, a JNK inhibitor can be used alone or in combination with two or more other JNK inhibitors. A p38 MAPK inhibitor can be used alone or in combination with two or more other p38 MAPK inhibitors. A JNK inhibitor can also be combined with a p38 MAPK inhibitor. In some embodiments, the at least one kinase inhibitor selected from the group consisting of a JNK inhibitor and a p38 MAPK inhibitor comprises one or more JNK inhibitors. In some embodiments, the at least one kinase inhibitor selected from the group consisting of a JNK inhibitor and a p38 MAPK inhibitor comprises one or more p38 MAPK inhibitors. In some embodiments, the at least one kinase inhibitor selected from the group consisting of a JNK inhibitor and a p38 MAPK inhibitor comprises a combination of at least one JNK inhibitor and at least one p38 MAPK inhibitor.

[0030] Furthermore, the at least one kinase inhibitor selected from the group consisting of a JNK inhibitor and a p38 MAPK inhibitor may not be able to penetrate the blood-brain barrier (BBB), and a delivery carrier or vehicle such as a viral vector, nanoparticle, exosome, or liposome may be used to deliver the at least one kinase inhibitor selected from the group consisting of a JNK inhibitor and a p38 MAPK inhibitor to the brain. Furthermore, the JNK inhibitor may be administered via a route that bypasses the BBB, such as intranasally or by direct injection into the brain. A preferred site in the brain is the cerebellum, more preferably including, but not limited to, at least one of cerebellar regions IV, V, and IX.

[0031] At least one kinase inhibitor selected from the group consisting of JNK inhibitors and p38 MAPK inhibitors is useful for suppressing cerebellar granule cell death caused by CASK gene mutations and can therefore be used as a therapeutic agent for CASK disorders. The most severe symptom of CASK disorders, including MICPCH syndrome, is cerebellar hypoplasia, and previous studies have shown that cerebellar granule cell death is central to this pathology. Therefore, if cerebellar granule cell death is suppressed by administering at least one kinase inhibitor selected from the group consisting of JNK inhibitors and p38 MAPK inhibitors, it is believed to be a therapeutic agent for cerebellar hypoplasia, i.e., CASK disorders, including MICPCH syndrome.

[0032] CASK abnormalities (also referred to as CASK-related disorders) include MICPCH syndrome, X-linked intellectual disability (XLID), and diseases caused by abnormalities in the CASK gene. Diseases caused by abnormalities in the CASK gene include, but are not limited to, one or more disorders selected from the group consisting of microcephaly, cerebellopontine hypoplasia (also referred to as cerebellar hypoplasia), facial abnormalities, intellectual disability, epilepsy, autism, nystagmus, optic atrophy, cleft palate, and cancer. In some preferred embodiments, the CASK abnormality includes MICPCH syndrome. CASK abnormalities can be diagnosed by identifying a pathogenic mutation in the CASK gene through genetic testing or by demonstrating an abnormality in the CASK gene through microarray karyotyping.

[0033] At least one kinase inhibitor selected from the group consisting of JNK inhibitors and p38 MAPK inhibitors, and therapeutic agents for CASK abnormalities containing such a kinase inhibitor as an active ingredient, can be used in subjects who are mammals, including humans, having an abnormality in the CASK gene (e.g., humans, mice, rats, dogs, cats, cows, horses, pigs, etc.). At least one kinase inhibitor selected from the group consisting of JNK inhibitors and p38 MAPK inhibitors, and therapeutic agents for CASK abnormalities containing such a kinase inhibitor as an active ingredient, may be used in non-human mammals having an abnormality in the CASK gene (e.g., heterozygous deficiency), or in humans suffering from a CASK abnormality, and are preferably used in humans suffering from a CASK abnormality.

[0034] 2. Pharmaceutical Composition The pharmaceutical composition of the present invention is a pharmaceutical composition containing at least one kinase inhibitor selected from the group consisting of a JNK inhibitor and a p38 MAPK inhibitor, and a pharmaceutically acceptable carrier.

[0035] JNK inhibitors and p38 MAPK inhibitors are as described above. In some embodiments, the at least one kinase inhibitor selected from the group consisting of JNK inhibitors and p38 MAPK inhibitors comprises one or more JNK inhibitors. In some embodiments, the at least one kinase inhibitor selected from the group consisting of JNK inhibitors and p38 MAPK inhibitors comprises one or more p38 MAPK inhibitors. In some embodiments, the at least one kinase inhibitor selected from the group consisting of JNK inhibitors and p38 MAPK inhibitors comprises a combination of at least one JNK inhibitor and at least one p38 MAPK inhibitor.

[0036] For example, the pharmaceutical composition can be, but is not limited to, a tablet, granule, capsule, powder, liquid, injection, suppository, patch, eye drop, or inhalant.

[0037] As the pharmaceutically acceptable carrier, various inorganic or organic carrier substances can be used. When the pharmaceutical composition is made into a solid preparation such as a tablet or granule, excipients, lubricants, binders, disintegrants, etc. can be used. When the pharmaceutical composition is made into a liquid preparation such as a liquid preparation or an injection, solvents, solubilizers, suspending agents, buffers, etc. can be used.

[0038] Furthermore, pharmaceutically acceptable additives such as substances that utilize the enhanced permeability of JNK inhibitors, antioxidants, preservatives, and coloring agents can be used as needed, and the additives can be selected depending on the administration method, administration site, etc. of the pharmaceutical composition.

[0039] When at least one kinase inhibitor selected from the group consisting of JNK inhibitors and p38 MAPK inhibitors is used as a pharmaceutical, it can be formulated with a pharmaceutically acceptable carrier and administered in one or more of a variety of dosage forms. Examples of such dosage forms include eye drops, ear drops, oral preparations, nasal preparations, injections (intrathecal, intraperitoneal, intravenous, intraarterial, intradermal, subcutaneous, and intramuscular), enteral preparations, suppositories, ointments, and patches. Each of these dosage forms can be prepared by a formulation method commonly known and commonly used by those skilled in the art.

[0040] The pharmaceutical composition of the present invention allows administration of at least one kinase inhibitor selected from the group consisting of a JNK inhibitor and a p38 MAPK inhibitor of the present invention at a daily dose of about 0.1 to 100 mg / kg body weight (preferably about 0.5 to 20 mg / kg body weight, more preferably about 0.1 to 10 mg / kg body weight) (when the pharmaceutical composition contains multiple kinase inhibitors, the total amount of the inhibitors).

[0041] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0042] Example 1 (1) Phenomena Observed in Cerebellar Granule Cells of CASK-Deficient Mice CASK heterozygous (female) and wild-type mice on postnatal day 6 (P6) and day 14 (P14) were perfusion-fixed with 4% PFA / PBS, followed by removal of the brain. After 24 hours of postfixation, the mice were immersed in 30% sucrose / PBS. After solution replacement, the mice were embedded in OCT compound. The embedded samples were sagittal sectioned at 30 μm thickness, mounted on glass slides, and air-dried for 30 minutes. The sections were stained with hematoxylin and eosin and observed under an optical microscope. Cerebellar granule cell density was assessed by counting cells in the granule cell layer inner than the Purkinje cell layer in the cerebellar lobule IV / V and lobule IX, and calculating the density per area. The two groups were analyzed using a Student's t test.

[0043] Figure 1A-C shows the results of observations of cerebellar granule cells in CASK-deficient mice.

[0044] As shown in Figure 1A, CASK heterozygous female mice (CASK+ / -) have reduced cerebellar size compared to CASK normal mice (CASK+ / +) on both postnatal days 6 (P6) and 14 (P14). Furthermore, as shown in Figure 1B and C, the density of cerebellar granule cells (CGCs) is reduced in CASK heterozygous female mice (CASK+ / -) compared to CASK normal mice (CASK+ / +).

[0045] Example 2 (2) Cell Culture Experiments with the Addition of Candidate Drugs Cerebellum was removed from CASK floxed mice 5-7 days after birth and divided into three sections using ultra-fine scissors. 1 mL of enzyme solution (1% trypsin, 0.1% DNase I) was added and incubated at room temperature for 3 minutes. After incubation, the supernatant was removed, and 2 mL of Neurobasal A medium (containing B-27 supplement, penicillin / streptomycin, and GlutaMAX supplement) containing 5% fetal bovine serum was added. After gentle mixing, the supernatant was removed. This process was repeated three times, after which 1 mL of pipetting solution (0.05% DNase I, 0.03% trypsin inhibitor, 2 mM MgCl2) was added and the mixture was slowly pipetted three times using a 1 mL pipette. The tip of a long-tipped Pasteur pipette was heated over a flame to further reduce the tip diameter, creating two pipettes (one thin and one thick), and the tissue was broken down by pipetting with the thick Pasteur pipette 2-3 times and then the thin Pasteur pipette 10 times.

[0046] The cell suspension was then passed through a 40μM cell strainer and centrifuged at 800 rpm for 4 minutes. The supernatant was removed and Neurobasal A medium containing 5% fetal bovine serum was added and the cells were counted. Round cover glasses coated with a coating solution (30mg / mL Poly-L-lysine, 10μg / mL laminin) were placed in the wells of a 24-well plate, and the cell suspension was seeded on the cover glasses at 2x10^5 cells / well. The day after the start of culture, the medium was replaced with Neurobasal A medium without fetal bovine serum, and lentivirus carrying the Cre recombinase gene was added at that time.

[0047] After 3 hours, four candidate drugs were added at concentrations of 0.01, 0.1, 1, or 10 μM. Each drug was dissolved in DMSO and stored at concentrations of 10 mM for SP600125, JNK-IN-8, and Doramapimod, and 20 mM for Tanzisertib. Three and five days after cell seeding, the medium was replaced with medium containing the drug at the respective concentrations. For medium replacement, 200 μL of medium was removed per well, followed by the addition of 250 μL of medium containing the candidate drug. Seven days after cell seeding, the culture supernatant was removed and cells were fixed in 4% PFA / 4% sucrose. Fixed cells were immunostained with an anti-NeuN antibody. Images were taken at three random locations per well using a confocal laser microscope. The number of NeuN-positive cells was calculated by taking the average of the three locations (N = 3 for each group). At the analyzed site, NeuN-positive cells mostly represent cerebellar granule cells. The two groups were tested using one-way ANOVA.

[0048] Example 2-1 SP600125 Figures 2 and 3 show the results of suppressing cerebellar granule cell death when SP600125 was added.

[0049] When Cre recombinase was introduced into cultured cerebellar granule cells prepared from CASK flox mice using a lentivirus to remove the CASK gene, the cerebellar granule cells died (lenti-Cre+DMSO). When the JNK inhibitor SP600125 was added to the cell culture medium, granule cell death was suppressed to some extent at concentrations of 10 μM or higher (lenti-Cre+SP600125), as shown in Figures 2 and 3. Furthermore, in the lenti-control+DMSO and lenti-control+SP600125 strains, Cre recombinase was not introduced into CASK flox cells (control strains were transfected with a lentivirus lacking Cre recombinase), and CASK was expressed, allowing the cerebellar granule cells to survive normally.

[0050] Example 2-2 JNK-IN-8 Figures 4 and 5 show the results of suppressing cerebellar granule cell death when JNK-IN-8 was added.

[0051] As in Example 2-1, when the JNK inhibitor JNK-IN-8 was added to the culture medium of CASK-deleted cerebellar granule cells, granule cell death was completely suppressed only when JNK-IN-8 was added at a concentration of 1 μM (lenti-Cre+JNK-IN-8), as shown in Figures 4 and 5. Note that in the lenti-control+DMSO and lenti-control+JNK-IN-8 cultures, cerebellar granule cells survived normally due to CASK expression.

[0052] [Example 2-3] Tanzisertib Figures 6 and 7 show the results of suppressing cerebellar granule cell death when tanzisertib was added.

[0053] As in Example 2-1, when the JNK inhibitor Tanzisertib was added to the cell culture medium of CASK gene-deleted cerebellar granule cells, granule cell death was suppressed to some extent at concentrations of 1 μM or higher of the JNK inhibitor (lenti-Cre+Tanzisertib), as shown in Figure 7. Note that in the lenti-control+DMSO and lenti-control+Tanzisertib cultures, cerebellar granule cells survived normally due to CASK expression.

[0054] [Example 2-4] Doramapimod The results of suppressing cerebellar granule cell death when doramapimod was added are shown in Figures 8 and 9. The two groups were tested by one-way ANOVA.

[0055] As in Example 2-1, when the JNK inhibitor Doramapimod was added to the cell culture medium of CASK gene-deleted cerebellar granule cells, granule cell death was suppressed to some extent at concentrations of the JNK inhibitor of 0.1 μM or higher (lenti-Cre+Doramapimod), as shown in Figures 8 and 9. Note that in the lenti-control+DMSO and lenti-control+Doramapimod cultures, cerebellar granule cells survived normally due to CASK expression.

[0056] Figure 10 shows the inhibitory effects on cerebellar granule cell death of the four JNK inhibitors (SP600125, JNK-IN-8, Tanzisertib, and Doramapimod) used in the experiment in Example 2. The concentration at which each JNK inhibitor had the greatest inhibitory effect is graphed.

[0057] Both JNK inhibitors were shown to suppress cerebellar granule cell death, suggesting their potential for preventing cerebellar hypoplasia in MICPCH syndrome. Addition of 1 μM JNK-IN-8 almost completely suppressed cell death, making them promising therapeutic agents for MICPCH syndrome.

[0058] Example 2-5 JNK-IN-7 Figure 11 shows the results of suppressing cerebellar granule cell death when JNK-IN-7 was added.

[0059] As in Example 2-1, when the JNK inhibitor JNK-IN-7 was added to the culture medium of CASK gene-depleted cerebellar granule cells, granule cell death was significantly suppressed when JNK-IN-7 was added at concentrations of 1 μM or higher (fCASK iCre vs. fCAK lenti), as shown in Figure 11. The two groups were tested by one-way ANOVA.

[0060] [Example 2-6] DB07268 Figure 12 shows the results of suppressing cerebellar granule cell death when DB07268 was added.

[0061] As in Example 2-1, when the JNK inhibitor DB07268 was added to the cell culture medium of CASK gene-deleted cerebellar granule cells, granule cell death was significantly suppressed when DB07268 was added at concentrations of 20 μM or higher (fCASK iCre vs. fCAK lenti), as shown in Figure 12. The two groups were tested by one-way ANOVA.

[0062] Example 3 (3) Local Cerebellar Injection of JNK Inhibitor JNK-IN-8 Six-day-old (P6) mice were placed on ice to sleep, their bodies taped to an ice pack, and their scalps incised along the midline, with the left and right sides secured with microclips. The lambda at the skull junction was set to 0, and the tip of the needle of a Hamilton Neurons syringe 700 filled with drug solution was inserted. The needle was moved 1.2 mm toward the torso and 1.0 mm to the left, inserted to a depth of 1.4 mm from the dura, and then moved back 0.1 mm to inject 3 μL at a depth of 1.3 mm.

[0063] The drug solution was prepared by dissolving JNK-IN-8 at a concentration of 10 mg / mL in 2% DMSO, 30% PEG300, 5% Tween 80, and 63% water. 2 μL of JNK-IN-8 solution was mixed with 1 μL of 0.4% trypan blue solution per mouse. After injection, the mice were sutured and warmed for 30 minutes before being returned to their mothers. Two weeks after drug injection, the mice were perfused and fixed, and granule cell density was measured.

[0064] FIG. 13 shows the recovery of cerebellar granule cell density when the JNK inhibitor JNK-IN-8 was injected locally into the cerebellum of CASK heterozygous deficient mice (CASK-KO).

[0065] The cerebellar granule cell density in regions IV / V and IX of CASK-normal (WT) and CASK-heterozygous mice was measured after injection of control (DMSO) or JNK inhibitor (JNK-IN-8) into the cerebellum. The cerebellar granule cell density in wild-type mice (WT) was set at 100%. In CASK-heterozygous mice (CASK-KO / DMSO), the cerebellar granule cell density was reduced to 61.598% in regions IV / V and 67.980% in region IX.

[0066] On the other hand, local cerebellar injection of JNK-IN-8 in CASK heterozygous mice (CASK-KO / JNK-IN-8) restored cerebellar granule cell density to 90.244% in cerebellar regions IV / V and 94.832% in cerebellar region IX, suggesting that local cerebellar injection of JNK-IN-8 may be effective in preventing cerebellar granule cell death in CASK-KO mice.

[0067] Example 4 (4) Experimental Study on Recovery of Cerebellar Motor Neuron Disorders by Injection of the JNS Inhibitor JNK-IN-8 in CASK Gene-Deficient Mouse Model The JNK inhibitor JNK-IN-8 was injected locally into the cerebellum of mice on postnatal day 6 (P6) as in Example 3 above, and the mice were maintained for two weeks. The mice were held by their tails at a distance of 7 cm from the tail and suspended for 30 seconds. A video camera positioned 10 cm above the ground was used to film the suspended mice, with the body positioned parallel to the ground so that the front and back legs were visible, to observe limb spasticity. The condition of the limbs after 30 seconds was evaluated for each individual based on the clasping score shown in Table 1.

[0068] Figures 14(A) and (B) show the recovery of limb spasticity when the JNK inhibitor JNK-IN-8 was injected locally into the cerebellum of CASK heterozygous deficient mice.

[0069] When control (DMSO) or JNK inhibitor (JNK-IN-8) was injected into the cerebellum of CASK-normal mice (WT), the average clasping score was 0.00, indicating no limb spasticity. CASK-deficient mice (CASK-KO / DMSO) showed severe spasticity, with an average clasping score of 3.285.

[0070] In contrast, when JNK-IN-8 was injected locally into the cerebellum of CASK heterozygous mice (CASK-KO / JNK-IN-8), the average glamping score was 0.7142, indicating an improvement in limb spasticity. ****: P<0.0001, ns: not significant. This suggests that local cerebellar injection of JNK-IN-8 is effective in improving limb spasticity.

[0071]

[0072] Example 5 (5) Gait Analysis by Ink Experiment The JNK inhibitor JNK-IN-8 was injected locally into the cerebellum of mice on postnatal day 6 (P6) as in Example 3, and the mice were housed for 2 weeks. CASK-normal mice (WT) and CASK-heterozygous mice (CASK-KO) were left in the laboratory for 30 minutes before the Gain Analysis by Ink experiment began. A 100 cm long, 8 cm wide, and 15 cm high transparent acrylic tunnel was placed under a 100 cm long, 10 cm wide piece of construction paper. The mouse was grasped by the scruff of the neck and its tail was held down with fingers to stabilize its body. Red paint was applied to the entire sole of its front paws, and blue paint was applied to the entire sole of its hind paws. The mouse, with the paint on its paws, was placed at the entrance of the tunnel and allowed to walk toward the exit, and its footprints were recorded on the construction paper.

[0073] In CASK-normal mice (WT), the vertical distance between right hind paw prints (step length) was 5.12 cm, and the horizontal distance from the line connecting the right hind paw steps to the left hind paw print (step width) was 2.41 cm. In CASK-heterozygous mice (CASK-KO / DMSO), the vertical distance between paw prints was 3.46 cm and the horizontal distance was 1.78 cm, indicating a smaller gait. The results in Figure 14 indicate that CASK-heterozygous mice (CASK-KO / DMSO) exhibit spasticity of the limbs, suggesting that the decreased vertical and horizontal distances between paw prints reflect spasticity.

[0074] On the other hand, when JNK-IN-8 was injected locally into the cerebellum of CASK heterozygous mice (CASK-KO / JNK-IN-8), the vertical distance between footprints was 4.18 cm and the horizontal distance was 2.17 cm, demonstrating a significant recovery of gait. This suggests that local cerebellar injection of NK-IN-8 may be effective in improving gait (data not shown).

Claims

1. A therapeutic agent for CASK abnormalities, comprising as an active ingredient at least one kinase inhibitor selected from the group consisting of JNK inhibitors and p38 MAPK inhibitors.

2. The therapeutic agent according to claim 1, wherein the CASK abnormality includes MICPCH syndrome.

3. The therapeutic agent according to claim 2, which is used for suppressing cerebellar hypoplasia in MICPCH syndrome.

4. A pharmaceutical composition for treating CASK abnormalities, comprising the therapeutic agent according to any one of claims 1 to 3 and a pharma- ceutical acceptable carrier.

5. A method for treating cerebellar hypoplasia due to CASK deficiency, using at least one kinase inhibitor selected from the group consisting of a JNK inhibitor and a p38 MAPK inhibitor.

6. A method for treating cerebellar hypoplasia due to CASK abnormality as described in claim 5, which comprises injecting into the cerebellum a pharmaceutical composition comprising at least one kinase inhibitor selected from the group consisting of a JNK inhibitor and a p38 MAPK inhibitor.

7. A method for suppressing cerebellar hypoplasia due to CASK abnormality, comprising administering a therapeutic agent for CASK abnormality described in any one of claims 1 to 3.

8. Use of at least one kinase inhibitor selected from the group consisting of a JNK inhibitor and a p38 MAPK inhibitor for the preparation of a pharmaceutical composition for the treatment of CASK abnormalities.

Images