Methods for treating tuberous sclerosis or epilepsy and compositions for use in such methods

Abstract

A method for treating tuberous sclerosis (TSC) or epilepsy is provided, comprising administering to a subject a compound of formula (I), formula (II), or formula (III):(I)(II)(III) or a pharmaceutically acceptable salt thereof (wherein X is a halogen). Also provided are compositions for use in a method for treating TSC or epilepsy, comprising administering to a subject in need of treatment a composition comprising a compound of formula (I), formula (II), or formula (III) as shown above. [Formula 1] JPEG2026510724000007.jpg61170

Description

[Technical Field]

[0001] This disclosure relates to methods for treating tuberous sclerosis (TSC) or epilepsy, and compositions for use in methods for treating TSC or epilepsy. [Background technology]

[0002] Tuberous sclerosis (TSC) is a genetic disorder caused by mutations in either the TSC1 or TSC2 gene, which encodes hamartin and tuberine, respectively. These two proteins work together to suppress mTOR signaling by inhibiting the small G protein Rheb, which directly activates mTOR. Overactivation of mTOR in TSC leads to the formation of hamartomas in multiple organs, including the heart, kidneys, lungs, skin, eyes, and brain. The symptoms of TSC are very diverse. Neuropathology is the leading cause of morbidity and mortality. Up to 90% of patients experience neurological symptoms, including seizures such as infantile spasms or status epilepticus, and other cognitive, psychiatric, or behavioral disorders. Among several neurological features, the most problematic but currently untreatable symptoms of TSC are neurocognitive disorders and neuropsychiatric disorders such as autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), learning disabilities and cognitive impairments, destructive behaviors and emotional problems. These features are collectively referred to as TSC-associated neuropsychiatric disorders (TAND). Many individuals with TSC exhibit at least one or more TAND symptoms throughout their lives. Individuals with TSC have a relatively high percentage (up to 40%) of ASD compared to the population, which is less than 2%. ADHD is also very common in TSC, with estimated prevalence rates of 21% to 50%. Approximately 44% to 64% of TSC patients have intellectual disabilities, and about one-third of children with TSC exhibit learning difficulties. Individuals with TSC also exhibit other behavioral and emotional problems, such as aggression (13%–58%), self-injurious behavior (27%–41%), anxiety (13%–48%), and depression (19%–43%). The diversity of these clinical findings suggests variability in the gene mutations involved in this disorder, which makes the treatment of TSC challenging.

[0003] Epilepsy is common throughout life in patients with TSC (approximately 90%). About 70% of these patients are classified as having refractory epilepsy, and these patients do not respond to current antiepileptic drugs (AEDs). Since loss-of-function mutations in TSC1 or TSC2 lead to constitutive activation of mTOR, mTOR inhibition is a primary treatment strategy in TSC management, including TSC-epilepsy. mTOR inhibitors such as sirolimus (rapamycin) and everolimus are approved by the US FDA for the treatment of TSC-associated epilepsy. However, only 40% of patients with refractory TSC epilepsy responded to treatment. Furthermore, because mTOR inhibitors directly affect cell proliferation and growth, there are concerns about their use, particularly regarding infants and young children ingesting them long-term. Currently, there are no available treatments for TSC-related neuropsychiatric disorders and sleep-related disorders. Furthermore, conventional antiepileptic drugs cause significant side effects. One-third of epilepsy patients do not respond to these drugs, and two-thirds of TSC patients do not show any therapeutic effect. The efficacy of conventional mTOR inhibitors for TSC-related refractory epilepsy is limited. In summary, safe and effective alternative therapies are needed for TSC, as well as for the treatment of TAND and TSC-related sleep disorders and epilepsy. [Overview of the Initiative]

[0004] As described above, one of the objectives of the present invention is to provide a safe and / or effective alternative therapy for the treatment of tuberous sclerosis (TSC), including TAND, and TSC-associated epilepsy. The present invention provides a novel method for treating TSCs by using adenosine analog compounds that are inhibitors of equilibrium nucleoside transporter 1 (ENT1), addressing the pathogenesis and functional recovery of TSCs.

[0005] In one aspect of the present invention, a method for treating tuberous sclerosis or epilepsy, wherein the subject requiring treatment is given formula (I), formula (II), or formula (III): [ka] A method comprising administering a compound, a pharmaceutically acceptable salt thereof, or a composition thereof (wherein X is a halogen). Another aspect of the present invention relates to a composition for use in a method of treating tuberous sclerosis or epilepsy, comprising administering a composition comprising a compound of formula (I), formula (II), or formula (III) as described above to a subject requiring treatment.

[0006] Preferably, the above compound is N 6 -[(3-halothien-2-yl)methyl]adenosine, N 6 -[(4-halothien-2-yl)methyl]adenosine, and N 6-[(5-halothien-2-yl)methyl]adenosine. More preferably, the above compound is N 6 -[(5-iodothien-2-yl)methyl]adenosine, N 6 -[(4-iodothien-2-yl)methyl]adenosine, N 6 -[(3-iodothien-2-yl)methyl]adenosine, N 6 -[(5-bromothien-2-yl)methyl]adenosine, N 6 -[(4-bromothien-2-yl)methyl]adenosine, N 6 -[(3-bromothien-2-yl)methyl]adenosine, N 6 -[(5-chlorothien-2-yl)methyl]adenosine, N 6 -[(4-chlorothien-2-yl)methyl]adenosine, and N 6 -[(3-chlorothien-2-yl)methyl]adenosine, and is selected from the group consisting of.

[0007] Preferably, the above compound is N 6 -[(2-halothien-3-yl)methyl]adenosine, N 6 -[(4-halothien-3-yl)methyl]adenosine, and N 6 -[(5-halothien-3-yl)methyl]adenosine, and is selected from the group consisting of. More preferably, the above compound is N 6 -[(2-iodothien-3-yl)methyl]adenosine, N 6 -[(4-iodothien-3-yl)methyl]adenosine, N 6 -[(5-iodothien-3-yl)methyl]adenosine, N 6 -[(2-bromothien-3-yl)methyl]adenosine, N 6 -[(4-bromothien-3-yl)methyl]adenosine, N 6 -[(5-bromothien-3-yl)methyl]adenosine, N 6 -[(2-chlorothien-3-yl)methyl]adenosine, N 6 -[(4-chlorothien-3-yl)methyl]adenosine, and N 6Selected from the group consisting of -[(5-chlorothien-3-yl)methyl]adenosine.

[0008] Preferably, the compound, a pharmaceutically acceptable salt thereof, or a composition thereof is administered orally, intravenously, intramuscularly, subcutaneously, intraperitoneally, or locally. Preferably, the composition further comprises a pharmaceutically acceptable carrier, excipient, or vehicle. Preferably, the epilepsy is TSC-associated epilepsy. Therefore, the present invention offers at least the following advantages: 1. The claimed method has better efficacy in treating cognitive impairment compared to vigabatrin and has no detectable side effects. 2. The claimed method can effectively treat TSC-associated neuropsychiatric disorders (TAND), sleep disorders caused by TSC, and epilepsy, including TSC-associated epilepsy. [Brief explanation of the drawing]

[0009] [Figure 1A] This figure shows the effect of J4 on cognitive impairment and anxiety-like behavior in Tsc2+ / - mice according to one embodiment of the present invention. Figure 1A: Experimental procedure for a novel object recognition test. Day 1 was the acclimatization period, with no objects placed; Day 2 was the training period, with two identical objects placed; and Day 3 was the experimental period, with one familiar object (object A) and one novel object (object B) placed for the mice to choose. [Figure 1B] Figure 1B: The time spent searching for object A (white bar) and object B (blue bar) was determined as a percentage of the total search time for four groups: WT / Veh, WT / J4, Tsc2+ / - / Veh, and Tsc2+ / - / J4, for both the training and experimental phases. [Figure 1C] Figure 1C: Upper panel, formula for calculating the discrimination index. Lower panel, discrimination index for the four groups. [Figure 1D]Figure 1D: Schematic diagram of the open-field testing facility. [Figure 1E] Figure 1E: Shows representative trajectories for each group. [Figure 1F] The time spent in the central arena (red rectangle), as clearly shown in the schematic diagram in Figure 1F (Figure 1E), was determined for four different groups. Data are presented as mean ± SEM. Unpaired t-tests, one-way ANOVA, and Tukey's post-hoc test were used, with p<0.05 and ns not significant. [Figure 2A] This figure shows the effect of J4 on brain microstructural abnormalities in Tsc2+ / - mice according to one embodiment of the present invention. Figure 2A: DKI region-based analysis was performed. The brain regions ACC, EC, CA3, and CA1 shown are indicated in coronal section (upper panel), horizontal section (middle panel), and sagittal section (lower panel). [Figure 2B] Figure 2B: Mean kurtosis was calculated for each brain region in each group. [Figure 2C] Figure 2C: DTI tract-based analysis was performed to examine the integrity of white matter structure, fornix, and frontal forceps. [Figure 2D] Figure 2D: Anisotropy ratios were determined for these two white matter regions for four groups. Abbreviations: ACC, anterior cingulate cortex; EC, entorhinal cortex; CA3, Ammon's horn 3; CA1, Ammon's horn 1. Data are shown as ±SEM. One-way ANOVA and Tukey's post-hoc test were used, with *p<0.05, ns not significant. [Figure 3A] This figure shows the effect of J4 on the size of dysplastic astrocytes in Tsc2+ / - mice according to one embodiment of the present invention. Figure 3A: Immunohistofluorescence staining of mouse coronal sections for GFAP and Iba1 was performed to visualize hippocampal CA1. Scale bar: 200 μm; inset, 20 μm. [Figure 3B] Figure 3B: GFAP immunoreactivity was compared between WT and Tsc2+ / - mice treated with the indicated vehicle or J4, with respect to GFAP-positive cells (upper panel), GFAP intensity (center panel), and area of ​​GFAP immunostaining (lower panel). [Figure 3C] Figure 3C: Iba1 expression was also compared across the four groups in terms of Iba1-positive cells (upper panel), Iba1 intensity (center panel), and area of ​​Iba1 immunostaining (lower panel). [Figure 3D] Figure 3D: We also considered the hippocampal CA3. [Figure 3E] GFAP intensity (Figure 3E) was determined for four different groups. [Figure 3F] The area of ​​GFAP immunoreactivity (Figure 3F) was determined for each of the four groups. [Figure 3G] The area of ​​Iba1 immunoreactivity (Figure 3G) was determined for four different groups. [Figure 3H] Figure 3H shows a schematic diagram of the posterior splenic (RSP) cortex of the corpus callosum, which has been further analyzed. [Figure 3I] Figure 3I: Triple labeling with GFAP, Iba1, and NeuN was performed to analyze the cell number and morphology of various types of nerve cells. [Figure 3J] The quantitative results for Iba1-positive cells (Figure 3J) were determined for each group. Data are shown as mean ± SEM. One-way ANOVA and Tukey's post-hoc test were used to determine the following: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns not significant. [Figure 3K] The quantitative results for NeuN-positive cells (Figure 3K) were determined for each group. Data are shown as mean ± SEM. One-way ANOVA and Tukey's post-hoc test were used to determine the following: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns not significant. [Figure 4A]This figure shows the effect of J4 on the epileptic seizure threshold in Tsc2+ / - mice according to one embodiment of the present invention. Figure 4A: Epileptic seizure susceptibility was determined in WT and Tsc2+ / - mice treated with J4, such as by five intraperitoneal injections of a vehicle or pentylenetetrazole (PTZ) at a dose of 40 mg / kg. The percentage of animals that showed a racine score of 4 or higher is shown for each injection. A two-sided Student's t-test was used to compare WT / Veh and Tsc2+ / - / Veh, with *p<0.05. [Figure 4B] Figure 4B: PTZ-induced epileptic seizure scores were further determined for four groups during each injection. Data are shown as mean ± SEM, and two-sided Student's t-tests were used to compare WT / Veh and Tsc2+ / - / Veh, with *p<0.05. [Figure 4C] Figure 4C: A comparison was conducted on the PTZ-induced epileptic seizure scores of the four groups at the time of the first injection. Data are shown as mean ± SEM. One-way ANOVA and Tukey's post-hoc test were used, p<0.05. [Figure 5A] This figure shows the effect of vigabatrin (VGB) on side effects in Tsc2+ / - mice according to a comparative example of the present invention. Figure 5A: A novel object recognition test was performed in WT and Tsc2+ / - mice treated with the vehicle, VGB, or J4 as shown. The time spent searching for object A or B, shown as a percentage of the total time, was determined for each group (top panel). The discrimination index was determined and shown for each group. Data are shown as mean ± SEM. Unpaired t-tests were used to compare search times between objects, and one-way ANOVA and Tukey's post-hoc test were used for multiple comparisons of discrimination indices, with the results being *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns not significant. * indicates significance after post-hoc testing compared to WT / Veh, and # indicates significance after post-hoc testing compared to Tsc2+ / - / Veh. [Figure 5B]Figure 5B: Weight was recorded for the group that was shown at the start of treatment (at 6 weeks of age). [Figure 5C] Figure 5C: Co-immunostaining of GFAP (red) and Iba1 (magenta) in cortical and hippocampal tissue from WT and Tsc2+ / - mice using vehicle, VGB, or J4 treatment as shown. [Figure 6A] This figure shows the effect of J4 on the survival of Tsc1CKO mice according to one embodiment of the present invention. Figure 6A: The survival curve shows the survival rate of Tsc1CKO mice treated with a vehicle or J4 as shown after gene deletion. [Figure 6B] Figure 6B: Western blot data of hippocampal tissue from the indicated groups were performed 7 days after gene deletion to visualize S6 and pS6 protein expression, and α-tubulin functioned as an internal control. [Figure 6C] Figure 6C: The ratio between pS6 and S6 was quantified from Western blot data 7 days after gene deletion. [Figure 6D] Figure 6D: Western blot data of hippocampal tissue from the indicated groups were performed 21 days after gene deletion to visualize S6 and pS6 protein expression, and α-tubulin functioned as an internal control. [Figure 6E] Figure 6E: Quantitative results of the ratio between pS6 and S6 from Western blot data were obtained 21 days after gene deletion. [Figure 6F] Figure 6F: Co-immunostaining of DCX (green) and PV (magenta) in the hippocampal dentate gyrus, as well as the results of enlarged immunostaining of the rectangles indicated for each group. [Figure 6G] Figure 6G: Quantitative bar graphs showing PV-positive cell density (left panel) and DCX-positive area (right panel). [Figure 6H] Figure 6H: Fluorescent immunofluorescence staining of GFAP, S100β, and Iba1 in cortical layer V for the indicated groups. [Figure 6I] Figure 6I: A bar graph showing the quantitative analysis of the displayed antibodies. [Figure 6J] Figure 6J: Nissl staining was performed on the cortex of the indicated groups. [Figure 6K] Figure 6K: Quantitative results of cell size in cortical layer V are shown. [Figure 6L] Figure 6L: Nissl staining was performed on the dentate gyrus hilum of the indicated group. [Figure 6M] Figure 6M: Quantitative results of cell size of dentate gyrus hilum cells. Data are shown as mean ± SEM. For multiple comparisons, one-way ANOVA and Tukey's post-hoc test were used, with *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and ns not significant. [Figure 7A] The effect of J4 on PTZ-induced epileptic seizures and gliosis is demonstrated according to one embodiment of the present invention. Figure 7A: PTZ was administered at a dose of 35 mg / kg to WT mice treated with the vehicle (Veh), J4, and vigabatrin (VGB) as shown. Racine scores were determined for each group from the 7th injection to the 21st injection (left panel). Racine scores were compared between the J4 group and the Veh group (right panel). [Figure 7B] Figure 7B: The percentage of Racine scores greater than 4 was determined for three groups (left panel), and compared separately between the J4 group and the Veh group (right panel). [Figure 7C] Figure 7C: Co-immunostaining of GFAP and Iba1 in the cortical regions of WT mice and WT mice treated with Veh, J4, and VGB as shown. [Figure 7D] Figure 7D: Co-immunostaining of GFAP and Iba1 in the hippocampal region of WT mice and WT mice treated with Veh, J4, and VGB as shown. Data are shown as mean ± SEM. For multiple comparisons, two-way ANOVA and Fisher's LSD post-hoc test were used, with *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and ns not significant. [Figure 8A]This figure shows the effect of J4 on sleep EEG in Tsc1CKO mice 22 days after gene deletion according to one embodiment of the present invention. Figure 8A: The stages of wakefulness, non-REM (NREM) sleep, and REM (REM) sleep in WT animals were determined by time-frequency analysis and image display for both electroencephalography (EEG) and electromyography (EMG). Wakefulness was characterized by low-amplitude EEG with mixed high-frequency components combined with high-amplitude EMG. NREM sleep was characterized by a relative increase in EEG amplitude consisting mainly of delta and theta frequency components combined with a low EMG tone. REM sleep was characterized by low-amplitude theta-based EEG combined with loss of muscle tone and occasional muscle spasms. [Figure 8B] The amount of wakefulness (Figure 8B) was determined during the dark and light phases for WT mice and Tsc1CKO mice treated with Veh or J4 as shown. Significant differences in the amount of wakefulness, NREM sleep, and REM sleep between vehicle-treated WT mice and J4-treated Tsc1CKO mice were shown as *p<0.05 and ¶p<0.05, respectively. These were analyzed for multiple comparisons using ANOVA and Holm-Bonferroni post-hoc tests. Data are shown as mean ± SEM. [Figure 8C] NREM sleep duration (Figure 8C) was determined during the dark and light phases for WT mice and Tsc1CKO mice treated with Veh or J4 as shown. Significant differences in wakefulness, NREM sleep, and REM sleep duration between vehicle-treated WT mice and J4-treated Tsc1CKO mice versus vehicle-treated Tsc1CKO mice were shown as *p<0.05 and ¶p<0.05, respectively. These were analyzed for multiple comparisons using ANOVA and Holm-Bonferroni post-hoc tests. Data are shown as mean ± SEM. [Figure 8D]REM sleep duration (Figure 8D) was determined during the dark and light phases for WT mice and Tsc1CKO mice treated with Veh or J4 as shown. Significant differences in wakefulness, NREM sleep, and REM sleep duration between vehicle-treated WT mice and J4-treated Tsc1CKO mice compared to vehicle-treated Tsc1CKO mice were shown as *p<0.05 and ¶p<0.05, respectively. These were analyzed for multiple comparisons using ANOVA and Holm-Bonferroni post-hoc tests. Data are shown as mean ± SEM. [Modes for carrying out the invention]

[0010] In one embodiment, a method for treating tuberous sclerosis (TSC) or epilepsy, wherein the subject is given formula (I), formula (II), or formula (III):

[0011] [ka] A method is provided comprising administering a compound of formula (III), a pharmaceutically acceptable salt thereof, or a composition thereof (wherein X is a halogen), where the compound of formula (III) is also referred to herein as "JMF 1907".

[0012] In another embodiment, the compound is N 6 -[(3-halothien-2-yl)methyl]adenosine, N 6 -[(4-halothien-2-yl)methyl]adenosine, and N 6 -[(5-halothien-2-yl)methyl]adenosine may be selected. Preferably, the above compound is N 6 -[(5-iodothien-2-yl)methyl]adenosine, N 6 -[(4-iodothien-2-yl)methyl]adenosine, N 6 -[(3-iodothien-2-yl)methyl]adenosine, N 6 -[(5-bromotien-2-yl)methyl]adenosine (also known as "JMF3464" or "J4"), N 6-[(4-bromotien-2-yl)methyl]adenosine, N 6 -[(3-bromotien-2-yl)methyl]adenosine, N 6 -[(5-chlorothien-2-yl)methyl]adenosine (also known as "JMF3818"), N 6 -[(4-chlorothien-2-yl)methyl]adenosine, N 6 -[(3-chlorothien-2-yl)methyl]adenosine, or a combination thereof.

[0013] In another embodiment, the compound is N 6 -[(2-halothien-3-yl)methyl]adenosine, N 6 -[(4-halothien-3-yl)methyl]adenosine, and N 6 -[(5-halothien-3-yl)methyl]adenosine may be selected. Preferably, the above compound is N 6 -[(2-iodothien-3-yl)methyl]adenosine, N 6 -[(4-iodothien-3-yl)methyl]adenosine, N 6 -[(5-iodothien-3-yl)methyl]adenosine, N 6 -[(2-bromotien-3-yl)methyl]adenosine, N 6 -[(4-bromotien-3-yl)methyl]adenosine, N 6 -[(5-bromotien-3-yl)methyl]adenosine, N 6 -[(2-chlorothien-3-yl)methyl]adenosine, N 6 -[(4-chlorothien-3-yl)methyl]adenosine, or N 6 -[(5-chlorothien-3-yl)methyl]adenosine, or a combination thereof. In one embodiment, the compound, a pharmaceutically acceptable salt thereof, or a composition thereof is administered orally, intravenously, intramuscularly, subcutaneously, intraperitoneally, or via a local route. [Examples]

[0014] Materials and methods animal The animals used in this study were handled in accordance with the guidelines of the University Committee on the Care and Use of Experimental Animals at Taipei Medical University (Taipei, Taiwan). Mice were housed in an air-conditioned vivarium with free access to food and water and a 12 / 12-hour light / dark cycle. Only male mice (2-3 months old) were used. Tsc2 + / - Knockout mouse model (B6;129S4-Tsc2) tm1Djk The Tsc1 knockout mouse model (Tsc1) was purchased from Jackson Laboratory (Bar Harbor, ME, USA). CKO ) is generated as follows: Tsc1 f / f -Tg(Camk2a-CreERT2 + First, transgenic mice possessing tamoxifen-inducible CreERT2 under the control of the Camk2a promoter (Tg(Camk2a-cre / ERT2), Jackson Laboratory) were developed, followed by (floxed) Tsc1 mutant mice in which conditional biallelic loxP was introduced (Tsc1 tm1Djk It is produced by crossbreeding with (Jackson Laboratory)

[0015] Drug administration Tsc2 + / - For mice, the dose of J4 administered was 0.06 mg / ml in drinking water containing 1% HPβCD. Administration was started at 6 weeks of age. The duration of treatment was 10 weeks. Tsc1 CKO For mice, the dose of J4 administered was 0.02 mg / ml in drinking water containing 1% HPβCD. Administration was started at 7-9 weeks of age. The duration of treatment was 3 weeks. Tamoxifen (concentration 20 mg / mL) was dissolved in 90% corn oil and 10% ethanol, and then administered intraperitoneally to mice aged 7-9 weeks for four consecutive days during the light phase (75 mg / kg, days 0-3) to activate CreERT2 and delete the Tsc1 gene in neurons. Two types of pentylenetetrazole (PTZ) kindling models were used. First, a PTZ kindling model using an alternate-day low-dose PTZ administration schedule was used. [1] In short, PTZ was prepared at a concentration of 4 mg / ml in sterile 0.9% (w / v) NaCl on the day of use and administered intraperitoneally at a dose of 40 mg / kg to induce epileptic seizures. PTZ was administered every other day when the mice were 9-10 weeks old. A total of 6 injections of PTZ were administered. Next, a similar kindling procedure was performed. To observe chronic epileptic seizures, when the mice were 7-8 weeks old, a total of 21 injections of PTZ at a dose of 35 mg / kg and a concentration of 3.5 mg / ml were administered every other day.

[0016] Brain slice specimens and immunohistochemical staining Mice were subjected to transcardiac perfusion with 10% formalin and then decapitated. After removal from the skull, the brain was post-fixed overnight in 10% formalin at 4°C. The fixed brain was dehydrated in 30% sucrose in 0.5M PB for 4 days and then embedded in OCT. 30 μm thick sections were obtained using a Leica CM1950 cryomicrotome (Leica Biosystems, Wetzlar, Hesse, Germany).

[0017] For immunofluorescence staining, slices were incubated overnight at 4°C with the desired antibody. After washing with PBS, the slices were incubated at room temperature for 1 hour with the corresponding Alexa Fluor dye-tagged secondary antibody. After washing three times with PBS, the tissue slices were placed on glass slides, mounted with anti-fading mounting medium (Vector Laboratories, Burlingame, CA, USA), and the cell nuclei were stained with Hoechst 33258 (Sigma-Aldrich, Missouri, USA). Images were acquired with a Leica STP6000 fluorescence microscope (Leica Biosystems), scanned with TissueGnostics (TissueGnostics GmbH, Vienna, Austria), and visualized with TissueFAXS & HistoFAXS (TissueGnostics GmbH). For quantification, six randomized regions (dimensions 200 × 200 μm) of the desired brain region were selected for each group. 2 The immunoreactivity or strength of each protein was selected from three different animals (N=3) and analyzed. The immunoreactivity or strength of each protein was determined using the "Measure" function of Fiji / ImageJ software (NIH, Bethesda, MD, USA; https: / / imagej.net / Fiji). Cell counts were counted using the "Analyze Particles" function of Fiji software and verified by the experimenter.

[0018] For Nissl staining, brain slices were first placed on glass slides. The glass slides containing tissue were placed in cresyl violet acetate solution for 5 minutes. After briefly rinsing the glass slides with PBS, they were dehydrated in stepwise alcohols (i.e., 50%, 75%, and 95% alcohol). Finally, the glass slides were purified in xylene and mounted with mounting medium. Images were acquired with TissueGnostics and visualized with TissueFAXS & HistoFAXS (TissueGnostics GmbH). For quantification, for each group, three randomized regions (dimensions 200 × 200 μm) of the desired brain region were selected. 2Cell counts were selected from three different animals (N=3) and analyzed. Cell counts were counted using the "Analyze Particles" function of Fiji software and verified by the experimenter.

[0019] PTZ-induced epileptic seizures Pentylenetetrazole (PTZ) (Sigma-Aldrich) was prepared on the day of use by dissolving it in sterile 0.9% (w / v) NaCl at a concentration of 3.5 or 4 mg / ml, and administered intraperitoneally at a dose of 35 or 40 mg / kg (body weight) to induce epileptic seizures. After PTZ injection, mice were placed in a clear observation cage for 30 minutes, and video was recorded for behavioral epileptic seizure scoring and quantification of epileptic seizure frequency. The severity and scoring of epileptic seizures were based on publicly available scoring criteria. [2] The Racine scale has been modified and is now concisely described as follows: 0, normal; 1, immobility and prone; 2, head nodding, spasms of the forelimb or hindlimb; 3, myoclonus reflex, tail raising; 4, standing up, chronic epileptic seizure, collapse; 5, tonic-clonic seizure, irregular jumping; 6, death.

[0020] Behavioral Test Before each behavioral test session, the mice were acclimatized in the behavioral chamber for 30 minutes to 1 hour. The novel object recognition (NOR) test consisted of three days, and an outline of the procedure is shown in Figure 1A. Day 1 was acclimatization, Day 2 was training, and Day 3 was the experiment. On Day 1, during acclimatization, the mice were allowed to freely explore an arena measuring 60 × 60 × 35 cm for 10 minutes. On the following day, during the training session, the mice were placed in the same arena with two identical objects (Object A) and allowed to acclimate to Object A for 10 minutes. On Day 3, during the experiment session, the mice were again placed in the same arena with one familiar object (Object A) and one new object (Object B). The mice were given 10 minutes to choose between the two objects. The discriminant index (DI) was calculated by dividing the time difference between Object A and Object B by the total time spent exploring Object A and Object B (Figure 1C). The open field (OFT) test was performed using day 1 of the non-reactive environment (NOR) test. Mice were allowed to freely explore the arena for 10 minutes without any objects. A central area measuring 29 × 29 cm was selected (Figure 1D), and the duration of the animals' stay in this area was observed. The movement trajectories of all behavioral tests were videotaped and analyzed using the open-source Matlab program OptiMouse. [3] .

[0021] MRI acquisition Magnetic resonance (MR) images were acquired using a 7 Tesla scanner (Bruker Biospec 70 / 30 USR, Ettlingen, Germany) with a 30 cm bore, and radio frequency pulses were transmitted using a linear volume coil. For the recovery of the radio frequency signal, a planar surface coil (T7399V3; Bruker Corp., Billerica, MA, USA) was placed on the head of each mouse. During each MRI session, the mice were anesthetized by inhalation of 3% isoflurane (Attane® Isoflurane, Minrad Inc., NY, USA) combined with 20% O2, 75% N2, and 5% CO2. The mice were secured in animal holders, and body temperature was maintained by placing a hot pad set to 37°C around their abdomens. A life monitoring system and pressure sensors (SA Instruments Inc., New York, NY, USA) were also placed under the abdomen of the mice to monitor their respiratory status. Respiratory rate remained stable, maintained at 20–40 breaths per minute.

[0022] Magnetic field uniformity is measured within a 7×7×7mm area encompassing the imaging slice. 3 For isotropic voxels, optimization was performed using a fast automated shimming technique (FASTMAP) with projection-aligned mapping along with a primary shim. Turbo spin echo (TSE) T2 images were acquired and slice positioning was confirmed [TR=2,500ms, TE=33ms, matrix size=256×256×15, field of view (FOV)=20×20mm]. 2 Voxel size = 0.08 × 0.08 × 0.4 mm 3[Slice thickness = 0.4 mm, 14 horizontal slices]. Diffuse kurtosis images were acquired using the DtiEpi SpinEcho sequence. TR = 3750 ms and TE = 31 ms, matrix size = 80 × 80 × 15 pixels, FOV = 20 × 20 mm 2 Slice thickness = 0.4 mm, 15 horizontal slices.

[0023] statistical analysis Regarding animal behavior, the time spent in the central arena for NOR discrimination index and OPF was measured for three groups: WT / Veh, WT / J4, and Tsc2. + / - / Veh, Tsc2 + / - Between / J4, we compared them using one-way ANOVA and Tukey's test for post-hoc analysis to determine statistical significance. For immunohistochemistry image analysis, both the mean GFAP+ cell count, Iba1+ cell count, and NeuN+ cell count, as well as immunoassay, were evaluated using one-way ANOVA and Tukey's test as a post-hoc analysis. One-way ANOVA and Fisher's LSD test as a post-hoc comparison were performed on the diffusion kurtosis (DKI) and diffusion tensor imaging (DTI) indices (MK for ROI-based analysis; FA for tract-based analysis) among the three groups. All of the above statistical analyses were performed using Prism version 8 (GraphPad Software, Inc., San Diego, CA, USA).

[0024] result J4 is Tsc2 + / - It improved cognitive impairment and anxiety-like behavior in mice. A novel object recognition (NOR) test (Figure 1A) was performed on Tsc2 treated with the vehicle. + / - Mouse (Tsc2 + / - While wild-type mice treated with Veh (WT / Veh) were found unable to distinguish between the familiar object A and the novel object B, wild-type mice treated with Vehicle (WT / Veh) or J4 (WT / J4) showed a significantly higher percentage of exploration time for the novel object B (Figure 1B). Tsc2 + / - The mice were treated with J4 (Tsc2+ / - When 0.06 mg / ml of 1% HPβCD was added to drinking water containing J4, the search time increased compared to object B. The discriminant index (DI) was calculated for each group. Tsc2 + / - The DI for the / Veh group was significantly lower than that for the other three groups (p<0.05, one-way ANOVA and Tukey's post-hoc test), and Tsc2 + / - The mice were shown to exhibit a lack of learning and memory function. Furthermore, treatment with J4 was able to reverse the deficiency (Figure 1C). These open-field behaviors were also analyzed by measuring the time spent in the central area for each group to determine anxiety-like behavior in the mice (Figure 1D). Trajectories for each group were monitored and analyzed (Figure 1E). From the trajectory and quantification results, Tsc2 + / - The / Veh group consists of WT / Veh and Tsc2 + / - Compared to / J4, the search time for the central region of the arena decreased (p<0.05, one-way ANOVA, Tukey's post-hoc test) (Figure 1F).

[0025] J4 is Tsc2 + / - We improved microstructural abnormalities in the brain of mice. Further diffusion kurtosis imaging (DKI) analysis was performed to determine the microstructural integrity of the mice and compare the three groups. Several brain regions were analyzed, and Tsc2 + / - We found that / Veh mice showed a decrease in mean kurtosis (MK) in the following regions: anterior cingulate cortex (ACC), entorhinal cortex (EC), Ammon's horn 1 (CA1), and CA3 (p<0.05, one-way ANOVA and Fisher's LSD post-hoc test) (Figure 2A). J4 treatment restored MK in these regions, suggesting that these brain regions are reversible upon treatment (p<0.05, one-way ANOVA and Fisher's LSD post-hoc test) (Figure 2B).

[0026] Next, diffusion tensor imaging (DTI) tract-based analysis was performed to depict structural connectivity within brain networks. Two white matter (WM) regions, the fornix and frontal forceps, represent axonal bundles connecting or linking gray matter regions such as the ACC and hippocampus (Figure 2C). The anisotropy ratio (FA) value is used to represent Tsc2 + / - We found that it decreased in / Veh mice and significantly increased in the fornix after J4 treatment (Figure 2D), but not with frontal forceps. Nevertheless, both DKI and DTI analyses were positive for Tsc2 + / - The / Veh mice showed abnormal brain microstructure in the gray matter (GM) and WM regions, respectively.

[0027] J4 is Tsc2 + / - The size of dysplastic astrocytes was reduced in mice. To identify possible cellular changes contributing to MK changes in MR imaging, immunofluorescence staining was performed using NeuN, GFAP, and Iba1 antibodies to visualize the number and morphology of neurons, astrocytes, and microglia, respectively. The hippocampal CA1 region (Figure 3A) was examined first. Tsc2 + / - We found that / Veh mice showed increased GFAP immunoassay and GFAP staining area compared to WT / Veh mice, but did not show an increase in GFAP-positive cells (Figure 3B). On the other hand, Tsc2 + / - / J4 mice showed a reduction in the area stained with GFAP, but not a decrease in GFAP intensity, suggesting that the treatment has the ability to reduce the size of GFAP-positive astrocytes and reduce astrocyte activation, but not a reduction in GFAP expression levels (Figures 3A and 3B).

[0028] Conversely, there were no significant changes in the number of Iba1-positive cells, Iba1 intensity, or Iba1 staining area, but Tsc2 + / - An increasing trend was observed in / Veh mice, and Tsc2 + / - A decreasing trend was observed in / J4 mice (Figure 3C). Further analysis of microglia using other markers is needed to confirm the effect of J4 on microglia.

[0029] Similarly, in the hippocampal CA3 subregion, the same effect of J4 on GFAP-positive astrocytes was observed (Figs. 3D-3F). Tsc2 + / - The morphology of Iba1-positive microglia in Tsc2 + / - / Veh mice (Fig. 3D, lower panel) was also studied, and no significant differences in the size and morphology of Iba1-positive cells were found compared to WT / Veh mice (Fig. 3G). Furthermore, the posterior corpus callosum (RSP) cortical region (Fig. 3H) was studied, and NeuN-positive and Iba1-positive cells in each group were determined (Fig. 3I). The number of Iba1-positive cells (Fig. 3J) and NeuN-positive cells (Fig. 3K) did not significantly change in the Tsc2

[0030] J4 increased the seizure threshold in Tsc2 + / - mice Tsc2 + / - mouse models do not appear to have spontaneous seizures, but Tsc2 + / - mouse models were hypothesized to have a lower seizure threshold when exposed to chemical challenges such as pentylenetetrazole (PTZ). This hypothesis was tested by observing seizure scores for each group using a PTZ-induced kindling protocol. PTZ was injected five times every other day at a subconvulsant dose (40 mg / kg, intraperitoneal), and WT / Veh was expected not to show signs of convulsive response or myoclonic reflex (stage 1). As expected, WT / Veh mice did not respond during the first and second injections of PTZ, whereas Tsc2 + / - / Veh mice showed an increase in the percentage of animals with Racine scores greater than 4 (Fig. 4A). At the first injection of PTZ, the seizure behavior of Tsc2 + / - / Veh mice had an average Racine score of 2.8, whereas the average Racine score of the WT / Veh group was much lower (Racine score = 1) (Fig. 4B). When compared to Tsc2 + / - / Veh mice, Tsc2 + / -The J4 mice showed milder seizure behaviors (Figure 4C), indicating that treatment with J4 can protect Tsc2 + / - mice from PTZ-induced convulsive behaviors.

[0031] Vigabatrin caused severe side effects in Tsc2 + / - mice. Next, the effect of J4 was compared with that of vigabatrin (VGB), an antiepileptic drug often used to treat TSC-related seizure attacks in Tsc2 + / - mice. Tsc2 + / - Mice treated with vigabatrin (Tsc2 + / - / VGB) did not show their NOR performance restored as in the case of J4 (Figure 5A). Also, vigabatrin caused significant weight loss in both WT and Tsc2 + / - mice (Figure 5B). Furthermore, the Tsc2 + / - / VGB group showed substantial increases in GFAP-positive astrocytes in the cortical region and increased Iba1 expression in the cortex and hippocampus, whereas these side effects were not seen in the Tsc2 + / - / J4 group (Figure 5C). These results indicated that J4 has better efficacy in treating cognitive impairment and no detectable side effects compared with vigabatrin.

[0032] J4 extended the survival of Tsc1 CKO mice. Next, whether J4 is effective against TSC-related seizure attacks was tested by using a TSC model Tsc1 CKO mouse model with spontaneous seizures. [4] . The results showed that J4 was effective in Tsc1 CKOIt was shown that mouse survival could be extended (Figure 6A). Western blotting was performed 7 days after tamoxifen-induced Tsc1 removal to determine pS6 and S6 levels. Vehicle and J4-treated groups were compared to WT mice. It was found that J4 treatment could reduce pS6 hyperactivation (Figures 6B and 6C). Surprisingly, J4-induced Tsc1 CKO The normalization of the elevated pS6 in mice disappeared when Tsc1 was deleted after 21 days (Figures 6D and 6E). Furthermore, in the J4 treatment group, Tsc1 CKO Compared to mice, the mice showed an increase in the double cortin (DCX) immunoreactive area in the dentate gyrus of the hippocampus where adult neurogenesis occurs, but no significant change was observed in parvalbumin (PV)-positive cells (Figures 6F and 6G). Furthermore, the inventors also found that J4 may exhibit neuroinflammation after epileptic seizures. CKO We also found that J4 inhibits proliferative GFAP-positive astrocytes in the V cortical region of mice (Figures 6H and 6I). Furthermore, we found that J4 reduces the size of pyramidal neurons (Figures 6J and 6K) and dentate gyrus hilum cells (Figures 6L and 6M) in the V cortical region. In summary, the results indicate that J4 transiently inhibits the abnormal activation of pS6 caused by Tsc1 loss, increases adult neurogenesis, reduces reactive astrocytes in the cortex, decreases the size of enlarged cells, and subsequently inhibits Tsc1 CKO This suggests that it may extend the shortened lifespan of mice.

[0033] J4 reduced PTZ-induced epileptic seizures and gliosis. To test whether J4 can be applied to epilepsy in general, a PTZ kindling model was used with a low-dose PTZ administration schedule every other day, and the effects of J4 and VGB were examined. For each injection, the epileptic seizure behavior of mice was videotaped and monitored to determine the Racine score. At the 16th, 17th, and 21st injections, J4 showed a significant difference compared to the VGB group. Surprisingly, VGB was found to show a higher mean Racine score compared to the Veh group (Figure 7A, left panel). Comparing the mean scores between Veh and J4 separately, J4 was found to significantly reduce the mean Racine score at each PTZ injection (Figure 7A, right panel). Similarly, the VGB group showed a higher percentage of epileptic seizure behavior with a Racine score greater than 4, while the J4 group showed a significantly lower percentage compared to the Veh and VGB groups (Figure 7B). Furthermore, dual labeling was performed using immunofluorescence staining for GFAP and Iba1 in the cortical region (Figure 7C) and hippocampal region (Figure 7D) of each group. The results demonstrated that, upon PTZ induction, the Veh and VGB groups showed severe gliosis, while the J4 group showed milder activation of GFAP and Iba1.

[0034] Tsc1 22 days after gene deletion CKO The effect of J4 on sleep EEG in mice. The effect of J4 on the modulation of electroencephalogram (EEG) signals during various sleep stages was further investigated. Time-frequency analysis of both EEG and EMG, as well as image display, determined the stages of wakefulness, non-REM (NREM) sleep, and REM (REM) sleep in WT animals (Figure 8A). Wakefulness was characterized by low-amplitude EEG with mixed high-frequency components combined with high-amplitude EMG. NREM sleep was characterized by a relative increase in EEG amplitude, mainly consisting of delta and theta frequency components combined with low EMG tones. REM sleep was characterized by low-amplitude theta-based EEG combined with loss of muscle tone and occasional muscle spasms. During the light phase, vehicle (Tsc1 CKO / Veh) or J4(Tsc1 CKOVehicle treatment using the procedure / J4) WT (WT / Veh) and Tsc1 CKO We found no significant difference in the amount of wakefulness between mice and Tsc1. CKO / Veh mice exhibit WT / Veh and Tsc1 during the dark period. CKO Compared to the wake duration of / J4 mice, a significant increase in wake duration was observed (Figure 8B). During the dark period, the WT / Veh group and Tsc1 CKO When compared to the NREM sleep duration of the / J4 group, Tsc1 CKO The amount of NREM sleep duration related to / Veh was found to be much shorter. No difference was observed among the three groups during the light phase (Figure 8C). REM sleep was observed in both the dark and light phases for WT / Veh and Tsc1 CKO When compared to the / J4 group, Tsc1 CKO A significant decrease was observed in the WT / Veh group (Figure 8D). WT / Veh and Tsc1 CKO / J4 vs Tsc1 CKO Significant differences in the duration of wakefulness, NREM sleep, and REM sleep in / Veh were, respectively * Data points are shown with p<0.05 and p<0.05. These were analyzed for multiple comparisons using ANOVA and Holm-Bonferroni post-hoc tests. Data are shown as mean ± SEM.

[0035] conclusion According to the results above, the J4 treatment was Tsc2 + / - The J4 treatment was shown to improve cognitive function and anxiety-like behavior in mice. Compared to vigabatrin, a commonly used AED, the J4 treatment showed improved Tsc2 saturation during pentylenetetrazole (PTZ)-induced epileptic seizures. + / - In mice, the treatment demonstrated efficacy in lowering the epileptic seizure threshold and reduced adverse effects. Furthermore, by incorporating imaging of diffusion kurtosis and diffusion tensor, which are non-invasive neuroimaging aspects, the J4 treatment was found to be effective in reducing Tsc2 + / - We found that it is possible to reverse microstructural abnormalities in the brain of mice and enhance connectivity. Furthermore, we found that Tsc1, a TSC model with spontaneous epileptic seizures, is a viable alternative therapy. CKOTesting of J4 in a mouse model showed that J4 treatment extended lifespan. Therefore, the present invention provides a novel alternative treatment for TSC to treat both TSC-related mental disorders and epilepsy. Thus, these results suggest that the compounds of this application are promising candidates for the treatment of TSC.

[0036] References JPEG2026510724000004.jpg59166

Claims

1. A method for treating tuberous sclerosis or epilepsy, wherein the subject requiring treatment is given formula (I), formula (II), or formula (III): 【Chemistry 1】 A method comprising administering a compound, a pharmaceutically acceptable salt thereof, or a composition thereof (wherein X is a halogen).

2. The aforementioned compound is N 6 -[(3-halothien-2-yl)methyl]adenosine, N 6 -[(4-halothien-2-yl)methyl]adenosine, and N 6 The method according to claim 1, selected from the group consisting of -[(5-halothien-2-yl)methyl]adenosine.

3. where the compound is N 6 -[(5-iodothien-2-yl)methyl]adenosine, N 6 -[(4-iodothien-2-yl)methyl]adenosine, N 6 -[(3-iodothien-2-yl)methyl]adenosine, N 6 -[(5-bromothien-2-yl)methyl]adenosine, N 6 -[(4-bromothien-2-yl)methyl]adenosine, N 6 -[(3-bromothien-2-yl)methyl]adenosine, N 6 -[(5-chlorothien-2-yl)methyl]adenosine, N 6 -[(4-chlorothien-2-yl)methyl]adenosine, and N 6 -[(3-chlorothien-2-yl)methyl]adenosine, and the method according to claim 2, which is selected from the group consisting of

4. The aforementioned compound is N 6 -[(2-halothien-3-yl)methyl]adenosine, N 6 -[(4-halothien-3-yl)methyl]adenosine, and N 6 The method according to claim 1, selected from the group consisting of -[(5-halothien-3-yl)methyl]adenosine.

5. The aforementioned compound is N 6 -[(2-iodothien-3-yl)methyl]adenosine, N 6 -[(4-iodothien-3-yl)methyl]adenosine, N 6 -[(5-iodothien-3-yl)methyl]adenosine, N 6 -[(2-bromotien-3-yl)methyl]adenosine, N 6 -[(4-bromotien-3-yl)methyl]adenosine, N 6 -[(5-bromotien-3-yl)methyl]adenosine, N 6 -[(2-chlorothien-3-yl)methyl]adenosine, N 6 -[(4-chlorothien-3-yl)methyl]adenosine, and N 6 The method according to claim 4, selected from the group consisting of -[(5-chlorothien-3-yl)methyl]adenosine.

6. The method according to claim 1, wherein the compound, a pharmaceutically acceptable salt thereof, or a composition thereof is administered by oral, intravenous, intramuscular, subcutaneous, intraperitoneal, or local route.

7. The method according to claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier, excipient, or vehicle.

8. The method according to claim 1, wherein the treatment of the tuberous sclerosis includes the treatment of tuberous sclerosis (TSC)-associated neuropsychiatric disorders (TAND).

9. The method according to claim 1, wherein the treatment of tuberous sclerosis includes treatment of sleep disorders caused by tuberous sclerosis.

10. The method according to claim 1, wherein the epilepsy is TSC-related epilepsy.

11. For the subject requiring treatment, use formula (I), formula (II), or formula (III): 【Chemistry 2】 A composition for use in a method of treating tuberous sclerosis or epilepsy, comprising administering a composition comprising a compound or a pharmaceutically acceptable salt thereof (wherein X is a halogen).

12. The aforementioned compound is N 6 -[(3-halothien-2-yl)methyl]adenosine, N 6 -[(4-halothien-2-yl)methyl]adenosine, and N 6 A composition for use according to claim 11, selected from the group consisting of -[(5-halothien-2-yl)methyl]adenosine.

13. The aforementioned compound is N 6 -[(5-iodothien-2-yl)methyl]adenosine, N 6 -[(4-iodothien-2-yl)methyl]adenosine, N 6 -[(3-iodothien-2-yl)methyl]adenosine, N 6 -[(5-bromotien-2-yl)methyl]adenosine, N 6 -[(4-bromotien-2-yl)methyl]adenosine, N 6 -[(3-bromotien-2-yl)methyl]adenosine, N 6 -[(5-chlorothien-2-yl)methyl]adenosine, N 6 -[(4-chlorothien-2-yl)methyl]adenosine, and N 6 A composition for use according to claim 12, selected from the group consisting of -[(3-chlorothien-2-yl)methyl]adenosine.

14. The aforementioned compound is N 6 -[(2-halothien-3-yl)methyl]adenosine, N 6 -[(4-halothien-3-yl)methyl]adenosine, and N 6 A composition for use according to claim 11, selected from the group consisting of -[(5-halothien-3-yl)methyl]adenosine.

15. The aforementioned compound is N 6 -[(2-iodothien-3-yl)methyl]adenosine, N 6 -[(4-iodothien-3-yl)methyl]adenosine, N 6 -[(5-iodothien-3-yl)methyl]adenosine, N 6 -[(2-bromotien-3-yl)methyl]adenosine, N 6 -[(4-bromotien-3-yl)methyl]adenosine, N 6 -[(5-bromotien-3-yl)methyl]adenosine, N 6 -[(2-chlorothien-3-yl)methyl]adenosine, N 6 -[(4-chlorothien-3-yl)methyl]adenosine, and N 6 A composition for use according to claim 14, selected from the group consisting of -[(5-chlorothien-3-yl)methyl]adenosine.

16. The composition for use according to claim 11, wherein the composition is administered by oral, intravenous, intramuscular, subcutaneous, intraperitoneal, or local route.

17. The composition for use according to claim 11, wherein the composition further comprises a pharmaceutically acceptable carrier, excipient, or vehicle.

18. The composition for use according to claim 11, wherein the treatment of tuberous sclerosis includes the treatment of tuberous sclerosis (TSC)-associated neuropsychiatric disorders (TAND).

19. The composition for use according to claim 11, wherein the treatment of tuberous sclerosis includes the treatment of sleep disorders caused by tuberous sclerosis.

20. The composition for use according to claim 11, wherein the epilepsy is TSC-related epilepsy.

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