Methods for treating sleep disorders and compositions for use therein

Abstract

A method for treating a sleep disorder in a subject in need thereof is provided, comprising administering to the subject a compound of Formula (I), (II), or (III), or a pharmaceutically acceptable salt thereof, wherein X is a halogen. Also provided is a composition for use in a method for treating a sleep disorder in a subject in need thereof, comprising administering to the subject a composition comprising a compound of Formula (I), (II), or (III) as shown above. JPEG2026503973000006.jpg59170

Description

[Technical Field]

[0001] The present disclosure relates to methods of treating sleep disorders and compositions for use in methods of treating sleep disorders. [Background technology]

[0002] Insomnia is one of the most common sleep problems, thought to affect 30-40% of the general population. Insomnia is a sleep problem that causes difficulty falling asleep. It can be poor sleep quality, inability to maintain good sleep (wakefulness), difficulty falling asleep, or, most commonly seen in teenagers, the feeling of not getting a full, deep sleep despite sleeping for a long time. Long-term insomnia lasts longer than a month, while short-term insomnia lasts for several days or weeks. Psychological stress, chronic pain, certain medications, lifestyle changes, caffeine, alcohol, and stress are the most common causes of insomnia. Over the past few decades, Alzheimer's disease (AD) has become the most common chronic neurodegenerative disease in the elderly, affecting 50 million people. The typical pathological features for diagnosing AD are the progressive extracellular aggregation of amyloid beta (Aβ) plaques and the accumulation of phosphorylated tau (p-tau) in the brain after postmortem examination. Excess neurotoxic Aβ undergoes conformational changes, oligomerization, and aggregation to form plaques, disrupting brain physiological functions. Tau is a protein that stabilizes microtubules in neuronal axons. When the positively charged microtubule-binding domain of tau is affected by hyperphosphorylation, it loses its positive charge and dissociates from microtubules. Dissociated tau protein further aggregates into paired helical filaments, which ultimately form insoluble neurofibrillary tangles (NFTs) in axons. Aβ plaques and NFTs are toxic to neurons, interfering with synaptic transmission and cellular function and ultimately leading to neuronal apoptosis. Neurotoxic Aβ plaques and tau tangles cause oxidative stress, neuroinflammation, mitochondrial dysfunction, and DNA damage, which lead to the symptoms of AD. In addition to cognitive impairment, sleep disturbances have been reported as a common symptom of AD, severely impacting patients and / or caregivers. Sleep disturbances include difficulty falling asleep, sleep fragmentation, circadian rhythm disruption, daytime sleepiness, and nighttime awakenings. Studies focusing on the relationship between sleep and pathological markers have shown that the level of cortical Aβ measured by PET scans correlates with reduced non-rapid eye movement (NREM) sleep, and reduced NREM sleep is associated with tauopathy in patients with early-stage AD. In general, Aβ and p-tau are also important factors in sleep disturbances in AD. Several studies have speculated that sleep disorders are directly associated with cognitive impairment. Patients with mild cognitive impairment (MCI) and AD exhibit a 40% decrease in the density of the K complex during sleep, which is the cause of cognitive impairment. Furthermore, questionnaire surveys have shown that 24.5% of patients with mild to moderate AD suffer from sleep disorders, and this proportion is likely to increase over the next 30 years. Therefore, sleep disturbances are a potential risk factor that may further accelerate the progression of AD. Nevertheless, there are no medications that effectively alleviate the symptoms of AD. Summary of the Invention

[0003] Based on the above reasons, the present invention provides a novel method for treating sleep disorders by using adenosine analogue compounds. In one aspect of the invention, a compound of formula (I), (II) or (III)

[0004] [ka] A method of treating a sleep disorder in a subject in need thereof, comprising administering to the subject a pharmaceutically acceptable salt thereof, or a composition thereof, wherein X is a halogen.

[0005] In another aspect of the invention, a composition for use in a method for treating a sleep disorder in a subject in need thereof, comprising administering to the subject a composition comprising a compound of formula (I), (II) or (III) as shown above. Preferably, 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. More preferably, the compound is selected from the group consisting of 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.

[0006] Preferably, 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. More preferably, the compound is selected from the group consisting of 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, N6 -[(4-chlorothien-3-yl)methyl]adenosine, and N 6 -[(5-chlorothien-3-yl)methyl]adenosine.

[0007] Preferably, the therapeutically effective amount of the compound is 0.5 to 15 mg / kg, preferably 1 to 12 mg / kg. Preferably, the compound, a pharmaceutically acceptable salt thereof, or a composition thereof is administered by oral, intranasal, intravenous, intramuscular, subcutaneous, intraperitoneal or topical route. Preferably, the composition further comprises a pharmaceutically acceptable carrier, excipient or vehicle. Preferably, the subject has insomnia or Alzheimer's disease (AD). Preferably, the insomnia is stress-induced insomnia, caffeine-induced insomnia, or a combination thereof.

[0008] Thus, the present invention provides at least the following advantages: 1. The present invention may show benefit in modulating disturbances of homeostatic sleep induced by AD, stress and / or caffeine. 2. The present invention can alleviate symptoms of AD, including sleep disorders, and slow the rate of their worsening. [Brief explanation of the drawings]

[0009]

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[0010] Data are expressed as mean ± SEM. Aβ + STZ vs. control * p<0.05 and *** p<0.001. Aβ+STZ vs. Aβ+STZ_Ent1(i), ## p<0.01 and ### p<0.001. NS=not significant.

[0011] Detailed Description In one embodiment, a compound of formula (I), (II) or (III) [ka] A method for treating schizophrenia, comprising administering to a subject a pharmaceutically acceptable salt thereof, or a composition thereof, wherein X is a halogen.

[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. Preferably, 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 (also known as "JMF3464" or "J4"), N 6 -[(4-bromothien-2-yl)methyl]adenosine, N 6 -[(3-bromothien-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. Preferably, the 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, or N 6 -[(5-chlorothien-3-yl)methyl]adenosine, or a combination thereof. In one embodiment, the therapeutically effective amount of the compound is 0.5 to 15 mg / kg, preferably 1 to 12 mg / kg. In one embodiment, the compound, a pharmaceutically acceptable salt thereof, or a composition thereof is administered orally, intranasally, intravenously, intramuscularly, subcutaneously, intraperitoneally, or topically. Embodiment 1

[0014] Materials and Methods In the present invention, Aβ aggregates (Aβ 1-42 Sporadic AD (sAD) mice were established by unilateral intrahippocampal (ih) microinjection of α-glucosamine (αG) and intracerebroventricular (icv) administration of streptozotocin (STZ), a glucosamine-nitrosourea compound, and used to evaluate the efficacy of the novel Ent1 inhibitor J4 in mitigating nitric oxide (NO), cleaved caspase 3, and phosphorylated H2A histone family member X (γ-H2AX) levels, nuclear DNA-dependent serine / threonine protein kinase (DNA-PKcs) activity, cholinergic neuron loss in the medial septum-Broca's diagonal band, and improving cognitive impairment. The effect of Ent1 inhibitor J4 on sAD-induced sleep disturbances was also investigated.

[0015] animal Ten-week-old male wild-type C57BL / 6 mice (BioLASCO Taiwan Co., Ltd.) weighing 25–28 g were used during the study. Mice were housed in plastic cages and individually housed in separate sound-proof chambers. Food and water were available ad libitum. Mice were maintained in an automatically controlled room at a constant temperature (23 ± 1°C), a relative humidity of 50–60%, and a 12:12 light / dark cycle. All procedures and animal care were performed in accordance with the Animal Care and Use Committee of National Taiwan University.

[0016] Electroencephalogram (EEG) implantation Mice were deeply anesthetized with zoletil (8.3 mg / kg, i.p., Virbac, Carros, France) and xylazine (7.4 mg / kg, i.p., Sigma-Aldrich) and placed in a stereotaxic apparatus. The scalp was incised, and the head was positioned to keep the bregma in a horizontal plane. The connective tissue was then wiped with sterile cotton until the bregma was clearly visible. To record EEG signals, custom-made wire-wrapped 30 AWG electrodes were inserted through the skull and positioned over the cortex of the frontal and occipital lobes. The cannula and EEG electrodes were then fixed to the skull with dental cement (Templon, GC Corporation, Tokyo, Japan).

[0017] EEG recording and sleep analysis After 5 days of recovery from surgery, mice were housed in individual chambers and connected to recording cables for 2 days for acclimatization before recording. Baseline data were obtained from the first 24 hours before drug treatment and used to compare data from the next recording, 24 days after the last drug administration. EEG signals were filtered and amplified 10,000 times using an amplifier (Coulbourn Instruments, Lehigh Valley, PA, USA; model V75-01). Every 12-second epoch from the 12-hour recording was manually scored using the data acquisition software ICELUS (MROpp, University of Michigan). Wakefulness was defined as three states: wakefulness (low EEG amplitude and higher frequency), non-rapid eye movement (NREM), high EEG amplitude, and delta power greater than theta power, and rapid eye movement (REM), low EEG amplitude accompanied by increased theta power.

[0018] Experimental procedure Animals were divided into sham control, vehicle control, STZ+Aβ 1-42 , STZ+Aβ 1-42 Mice were randomly divided into four groups: STZ + Aβ, ENT1(i), and sham group. Mice in the sham group received 0.9% PFS into the ventricles and dorsal hippocampus (dHPC; 1 μl per injection site). 1-42 In the group, STZ (3 mg / kg) was injected intravenously on days 7 to 10, and Aβ 1-42( Mice were injected with ENT1 inhibitor (6 mg / kg) or its vehicle (1% HPβCD) for 18 days (days 7-24). Fourteen days after the last intravenous and intravenous injections of ENT1 inhibitor, along with oral administration of the ENT1 inhibitor, mice were subjected to assessment of locomotor activity and anxiety levels using the open field test (OFT). Cognitive function was assessed using the Morris water maze (MWM) and novel object recognition (NOR) tasks. EEG signals were recorded to investigate changes in sleep-wake activity. Immediately after behavioral assays and sleep recordings, mice were sacrificed and used for biochemical studies and immunofluorescence assays (IFA).

[0019] To examine the effects of the Ent1 inhibitor J4 on physiological sleep-wake activity, three different doses of the Ent1 inhibitor J4, 1, 6, and 12 mg / kg, were orally administered at either the onset of the dark phase or the onset of the light phase, and sleep-wake activity was recorded for 24 hours. To determine the effect of the Ent1 inhibitor J4 on caffeine-induced insomnia, caffeine was injected i.p. at the onset of the light phase, and three different doses (1, 6, and 12 mg / kg) of the Ent1 inhibitor J4 were orally administered 3 hours before the light phase, and sleep-wake activity was recorded for 24 hours. To evaluate the effect of the Ent1 inhibitor J4 on stress-induced insomnia, cage exchange was performed at the beginning of the light phase, and three different doses of the Ent1 inhibitor J4 (1, 6, and 12 mg / kg) were orally administered 3 hours before the light phase, and sleep-wake activity was recorded for 24 hours.

[0020] Novel object recognition task (NOR task) This behavioral task was performed in a white acrylic open box (40 × 40 × 45 cm) and was divided into three phases: habituation, training, and testing. On the first day, mice were allowed to freely explore the apparatus (used as the OFT) for 15 min to familiarize themselves with the surroundings. During the habituation phase, no objects were placed in the box. 24 h later, two identical objects (A1 and A2) were placed diagonally opposite each other and allowed to explore for 10 min. After the training phase, mice were returned to their home cages and allowed to rest within the intertrial interval (90 min) before the test phase. To assess the mice's memory ability, one of the familiar objects was replaced with a novel object (B) and allowed to explore for 5 min to acquire short-term memory (STM). A long-term memory test was performed 24 h after training, during which mice explored the familiar object A and another novel object C for 5 min. The objects and apparatus were wiped with 20% ethanol solution to avoid the presence of odor. Exploratory behavior was defined as sniffing an object from a distance of <2 cm or touching the object with the nose or forepaws. The discrimination index (DI) was calculated for each mouse and expressed as the ratio of the time spent on the familiar object (A) minus the time spent on the novel object (B or C), divided by the total exploration time for both objects (DI = ([B or C] - A) / ([B or C] + A)).

[0021] Morris Water Maze (MWM) The task took place in a circular pool (153 cm in diameter) filled with water (25 ± 1°C). The water was mixed with nontoxic white paint to turn the clear water opaque. The pool was divided into four quadrants. An escape platform (10 cm in diameter) was placed in the fourth quadrant (target quadrant) and submerged 2 cm below the water surface. Visual cues were attached to the edge of the pool to orient the mice. During the training phase, all mice were given three trials per session over five consecutive days (days 19, 20, 21, 22, and 23), with each trial requiring 120 s of searching for the hidden platform. After climbing onto the platform, the mice remained on the platform for 30 s before returning to their home cage. If they failed to find the platform, they were gently guided away and allowed to stay on the platform for 30 s. 24 h after the last training trial, a probe trial (day 24) was conducted by removing the escape platform from the target quadrant and allowing the mice to swim freely for 120 s. Escape latency, the time taken to reach the platform during the training phase, was assessed. Video tracking software (EthoVision XT version 14.0.1322, Noldus Information Technology, The Netherlands) measured the time spent in the target quadrant and the number of crossings of the original platform location during the probe trial.

[0022] result Characteristics of sAD established by streptozotocin (STZ) and Aβ After four unilateral intravenous microinjections of Aβ aggregates over four consecutive days and intravenous STZ administration, we observed characteristics of sAD on day 14. The accumulation of Aβ plaques in mice was measured by optical density after intravenous STZ and intravenous Aβ administration. Compared with control mice treated with PFS (Figure 1A), Aβ plaques were widely distributed in the CA1, CA3, and hilus of the hippocampus, as shown in Figure 1B. A comparison by unpaired Student's t-test revealed a statistically significant difference in amyloid beta deposition (Aβ-positive area / tissue area ratio) between the control group (≈0 pixels) and the group receiving intravenous STZ and intravenous Aβ (0.045 ± 0.011 pixels; p < 0.001 vs. control), as shown in Figure 1C.

[0023] Fourteen days after four doses of icv-STZ and ih-Aβ, we measured the levels of tau protein phosphorylated at residue Ser404. The results showed that the ratio of phosphorylated tau to total tau in the hippocampus increased significantly from 0.81 ± 0.08 in the control group to 2.27 ± 0.31 (p < 0.001, Figures 1D and 1E). However, the total tau protein level in the hippocampus did not change after treatment with icv-STZ and ih-Aβ (Figure 1F). These results indicate that four doses of icv-STZ and ih-Aβ can establish the accumulation of Aβ and phosphorylated tau protein in the brain, simulating sAD in mice.

[0024] Effect of Ent1 inhibitor J4 on cholinergic neuronal loss in the medial septum-diagonal band of Broca (MSDB) after icv-STZ and ih-Aβ Mice treated with icv-STZ and ih-Aβ showed a significant reduction in the number of choline acetyltransferase (ChAT)-positive neurons in the MSDB. The number of ChAT-positive neurons in the MSDB was 21.13 ± 0.06 (vs. control, p < 0.001), compared with 60.11 ± 2.98 neurons in the control group (Figures 2A, 2B, 2E). In sAD mice treated with the Ent1 inhibitor J4, cholinergic neuron loss was clearly prevented, with the number of cholinergic neurons in the MSDB maintained at 51.11 ± 1.15 (vs. Aβ + STZ, p < 0.001, Figures 2D and 2E). Because J4 was dissolved in a vehicle of 1% 2-hydroxypropyl-beta-cyclodextin (HPβCD), we also determined whether 1% HPβCD affected cholinergic neuronal loss. The results showed that HPβCD had no effect, as shown in Figures 2C and 2E. This result suggests that increasing extracellular adenosine levels by ENT inhibitors is beneficial to the basal forebrain cholinergic system.

[0025] Effect of Ent1 inhibitor J4 on recognition memory impairment in icv-STZ and ih-Aβ-induced sAD mice Based on the numerous abnormalities that occurred in the hippocampus and the cholinergic neuronal loss in the MSDB, behavioral assays, the novel object recognition (NOR) test and the Morris water maze (MWM) test, were used to determine whether cognitive function was impaired. As sAD was established by icv-STZ and ih-Aβ, short-term and long-term memory were assessed in the first and second test phases of the NOR study described in the Methods section that follows.

[0026] See Figure 3. Results showed that icv-STZ- and ih-Aβ-induced sAD mice were unable to discriminate between familiar and novel objects and spent more time exploring the familiar object compared with their control littermates. The discrimination indices obtained from sAD mice were −0.27 ± 0.12 (p < 0.01 vs. control, one-way ANOVA with post-hoc comparison) for short-term memory (STM) and −0.2 ± 0.06 (p < 0.05 vs. control, one-way ANOVA with post-hoc comparison) for long-term memory (LTM), which were significantly reduced compared with those obtained from controls for STM (0.27 ± 0.08) and LTM (0.21 ± 0.15). Meanwhile, oral administration of the Ent1 inhibitor J4 significantly improved the discrimination ability between familiar and novel objects in these sAD mice. The discrimination index obtained from sAD mice receiving the Ent1 inhibitor J4 was 0.49 ± 0.1 in STM (p < 0.001 vs. Aβ + STZ, one-way ANOVA with post hoc comparisons) and 0.62 ± 0.11 in LTM (p < 0.001 vs. Aβ + STZ, one-way ANOVA with post hoc comparisons). Oral administration of the vehicle HPβCD to control mice did not alter the discrimination index, regardless of STM or LTM. Furthermore, HPβCD vehicle did not significantly improve the decline in discrimination index obtained from sAD mice. These results clearly demonstrate that J4 prevents cognitive decline in icv-STZ- and ih-Aβ-induced sAD mice and suggest that the Ent1 inhibitor J4 improved recognition memory in sAD mice.

[0027] Effect of Ent1 inhibitor J4 on spatial memory impairment in icv-STZ and ih-Aβ-induced sAD mice Spatial memory learning was also determined by heatmaps of swimming trajectories in the Morris Water Maze (MWM) and time spent in the second quadrant with or without a hidden platform.

[0028] First, see Figure 4A. Escape latency, the time required to reach the platform, was assessed during the training phase. The results showed that mice treated with ih-Aβ and icv-STZ took significantly longer to reach the hidden platform than their control littermates on days 2, 4, and 5, indicating learning impairment. Interestingly, treatment with the Ent1 inhibitor J4 significantly improved the ability of sAD mice to find the hidden platform between days 2 and 5. Oral administration of HPβCD had no effect on escape latency in sAD mice. See Figure 4B. In the probe test, in which the hidden platform was removed, control mice showed a preference to stay in the target quadrant (quadrant 2), where the hidden platform was located during the training session. Mice treated with HPβCD showed no change in their preference to stay in quadrant 2 in the probe test. Mice treated with ih-Aβ and icv-STZ lost their preference to stay in quadrant 2, indicating spatial memory impairment, whereas J4 was able to reverse memory impairment in sAD mice. See also Figure 4C. The time spent in the target quadrant by sAD mice was significantly reduced to 14.04 ± 2.35 seconds compared to 31.52 ± 3.08 seconds obtained from control mice (sAD vs. control, p<0.01). Administration of the vehicle HPβCD did not improve the spatial memory of sAD mice. The Ent1 inhibitor J4 significantly improved the time spent in the target quadrant to 37.52 ± 5.75 seconds compared to that obtained from untreated sAD mice (p<0.001 vs. sAD mice). However, there was no significant change between control mice, sAD mice, and sAD mice treated with either vehicle or J4, suggesting that the Ent1 inhibitor J4 improved the spatial memory of sAD mice.

[0029] Effect of Ent1 inhibitor J4 on the expression of NO in sAD mice Treatment of mice with icv-STZ and ih-Aβ induces oxidative stress, which subsequently leads to DNA damage and neuronal apoptosis, as observed in the loss of cholinergic neurons in MSDB (see Figure 5). NO levels were assessed by the amount of nitrite. Nitrite concentrations significantly increased in the hippocampus after treatment with icv-STZ and ih-Aβ from 19.56 ± 1.72 μM and 18.04 ± 1.62 μM obtained in control and vehicle HPβCD, respectively, to 27.82 ± 2.23 μM (p < 0.05 vs. control or vehicle HPβCD). sAD mice treated with J4 showed attenuated nitrite levels to 18.33 ± 1.45 μM compared with those in sAD mice alone (p < 0.01), indicating that the Ent1 inhibitor J4 prevented oxidative stress in sAD.

[0030] Effects of Ent1 inhibitor J4 on DNA damage and apoptosis in sAD mice Abnormal increases in NO in the hippocampus may further lead to DNA damage and cell apoptosis (see Figure 6A). The results show that when sAD was established after treatment with icv-STZ and ih-Aβ, the ratio of γ-H2AX / α-tubulin, an indicator of DNA double-strand breaks (DSBs), was statistically significantly increased in the hippocampus. The ratio increased from 0.14 ± 0.03 and 0.2 ± 0.03 obtained from control and vehicle HPβCD, respectively, to 0.58 ± 0.06 (p < 0.001). Oral gavage administration of the Ent1 inhibitor J4 significantly attenuated the ratio of γ-H2AX / α-tubulin in the hippocampus to 0.37 ± 0.06 (p < 0.05 vs. sAD mice).

[0031] See also Figure 6B. Similar observations were found in the expression of cleaved caspase 3, an indicator of cell apoptosis. The cleaved caspase 3 / α-tubulin ratio increased to 0.69 ± 0.03 from 0.3 ± 0.06 and 0.25 ± 0.04 obtained from control and vehicle HPβCD, respectively (p < 0.001). Meanwhile, oral gavage administration of the Ent1 inhibitor J4 significantly reduced the cleaved caspase 3 / α-tubulin ratio in the hippocampus to 0.69 ± 0.03 (p < 0.001 vs. sAD mice). Collectively, enhancing extracellular adenosine by the Ent1 inhibitor J4 can alleviate DNA damage and apoptosis in sAD mice.

[0032] Effect of Ent1 inhibitor J4 on DNA-PKcs activity in sAD mice Following the increase in DNA DSB markers and apoptosis in sAD mice, we next assessed the activity of DNA-PKcs, a key enzyme mediating the NHEJ pathway that repairs DNA double-strand breaks. See Figure 6C for further details. The active form of DNA-PKcs is determined by phosphorylation of Thr2609. Western blot analysis revealed that the ratio of phosphorylated DNA-PKcs / DNA-PKcs decreased from 1.05 ± 0.13 and 1.02 ± 0.05 obtained from control and vehicle HPβCD, respectively, to 0.72 ± 0.06 (p < 0.05) after treatment with icv-STZ and ih-Aβ. Administration of the Ent1 inhibitor J4 significantly increased the ratio of phosphorylated DNA-PKcs / DNA-PKcs to 1.02 ± 0.05 (p < 0.05 vs. sAD mice). However, the level of total DNA-PKcs did not change between these groups.

[0033] Effect of Ent1 inhibitor J4 on sAD-induced sleep disturbance Because sleep disturbances are commonly observed in AD patients, we determined changes in sleep-wake activity and sleep structure after administration of icv-STZ and ih-Aβ, as well as the effects of J4. Results revealed that NREM sleep during the first 3 h of the dark phase (Zeitgeber time [ZT] 13–15) significantly increased from 11.04 ± 3.92% in controls to 38.83 ± 2.54% in icv-STZ and ih-Aβ-induced sAD mice (p<0.001, one-way ANOVA with post hoc comparisons, Figures 7A and 8A). Meanwhile, NREM sleep during the light phase of the subsequent light:dark cycle (ZT 3–5) was suppressed from 57.69 ± 3.56% in controls to 35.16 ± 3.52% (p<0.05, one-way ANOVA with post hoc comparisons, Figures 7A and 8B). Furthermore, REM sleep from ZT'1 to ZT'11 significantly decreased from 10.7 ± 0.69% in controls to 3.04 ± 0.49% (p < 0.001, one-way ANOVA with post hoc comparisons, Figures 7B and 8C). Wakefulness showed a mirror effect in response to sleep changes. Wakefulness significantly decreased from 88.31 ± 4.24% to 59.02 ± 2.9% (p < 0.05, one-way ANOVA with post hoc comparisons) between ZT'13 and ZT'15 in sAD mice (Figure 8D), and significantly increased from 23.81 ± 3.11% to 46.68 ± 2.81% (p < 0.05, one-way ANOVA with post hoc comparisons) between ZT'3 and ZT'9 (Figures 7C and 8E).

[0034] Oral administration of J4 reversed the enhancement of NREM sleep from ZT13 to ZT15 to 17.87 ± 3.96% (p < 0.05 vs. Aβ + STZ, one-way ANOVA with post hoc comparisons, Figures 7D and 8A) and normalized the suppression of NREM sleep from ZT3 to ZT5 to 63.75 ± 4.11% (p < 0.05 vs. Aβ + STZ, one-way ANOVA with post hoc comparisons, Figures 7D and 8A). J4 blocked the sAD-induced suppression of REM sleep from ZT1 to ZT11, with the amount of REM sleep after J4 administration being 8.91 ± 0.56% (p < 0.05 vs. Aβ + STZ, one-way ANOVA with post hoc comparisons, Figures 7E and 8C). J4 also reversed the decrease in wakefulness at ZT13–15 and the enhancement of wakefulness at ZT'3–9 by 82.12 ± 4.16% (p < 0.05 vs. Aβ + STZ, one-way ANOVA with post hoc comparisons, Figures 7F and 8D) and 28.38 ± 3.0% (p < 0.05 vs. Aβ + STZ, one-way ANOVA with post hoc comparisons, Figures 7F and 8E), respectively.

[0035] Analysis of sleep architecture revealed that the reduction in NREM sleep during the light phase after treatment with icv-STZ and ih-Aβ was primarily due to a decrease in NREM bout duration, whereas the number of NREM bouts increased (Figures 8F and 8G). Application of the Ent1 inhibitor J4 successfully prevented the reduction in NREM bout duration and reversed the enhancement in the number of NREM bouts in sAD mice (Figures 8F and 8G). The reduction in REM sleep during the light phase was due to a decrease in both REM bout duration and number in sAD mice, and J4 blocked these suppressions (Figures 8H and 8I). Furthermore, sAD mice showed increased transitions between wakefulness states, suggesting sleep fragmentation, which J4 corrected (Figure 8J).

[0036] Consideration Sleep problems are becoming increasingly problematic in AD. sAD is the most common neurodegenerative disease, accompanied by neuronal apoptosis, cognitive decline, and sleep disorders. Results from embodiments of the present invention revealed significant changes in sleep architecture in Aβ + STZ-induced sAD mice. The affected sleep patterns showed that NREM sleep, REM sleep, and wakefulness were not in line with normal sleep homeostasis and were accompanied by shorter sleep durations. However, there were no significant differences in light-phase transitions. Although the transitions were inconclusive, a tendency toward sleep fragmentation could be observed in Aβ + STZ-induced sAD mice. It can be concluded that the changes in sleep-wake activity in icv-STZ and ih-Aβ-induced sAD mice are similar to the sleep disorders in AD patients.

[0037] Sleep disorders and AD even show a bidirectional relationship. Reduced sleep duration may increase the production of pathological features of AD, and abnormal aggregation of neurotoxic proteins may lead to neuronal damage and altered sleep patterns. In addition to its impact on the progression of AD, several studies have investigated the relationship between sleep disruption and memory decline. Impairments in hippocampal sharp wave ripples and theta rhythm during NREM and REM sleep, respectively, have been correlated with impairments in memory consolidation and storage.

[0038] Our results show that treatment with the Ent1 inhibitor J4 normalized the levels of nitric oxide, cleaved caspase 3, and phosphorylated H2A histone family member X (γ-H2AX) and increased the activity of nuclear DNA-dependent serine / threonine protein kinase (DNA-PKcs) via the non-homologous end joining (NHEJ) pathway, which repairs DNA double-strand breaks. J4 also attenuated the loss of cholinergic neurons in the medial septum-Broca's diagonal band, further improving cognitive impairment. Furthermore, sAD mice showed increased NREM sleep during the dark phase and decreased NREM and REM sleep during the light phase, indicating that elevated extracellular adenosine is beneficial for homeostatic sleep. In other words, treatment with the Ent1 inhibitor in the present invention normalized the disturbances in sleep patterns in sAD mice, suggesting that the increased adenosine caused by the Ent1 inhibitor has a positive effect on the regulation of homeostatic sleep. Adenosine is a sleep-promoting substance, and blocking Ent1 increases extracellular adenosine. Therefore, it can be speculated that the enhanced binding between adenosine receptors and adenosine further regulates sleep in the CNS. In conclusion, the Ent1 inhibitor J4 may have potential for the treatment of sAD.

[0039] Embodiment 2 Materials and Methods Animal and EEG implants This study used male C57BL / 6 mice (6–8 weeks old; BioLASCO Taiwan Co., Ltd.) and GAD67-GFP mice. The original strain of GAD67-GFP mice was B6 (provided by Dr. MY Ming of National Taiwan University). Mice were anesthetized with zoletil (10 mg / kg, Carros, France) and xylazine (12 mg / kg, Sigma-Aldrich, USA). Male C57BL / 6 mice were implanted with two EEG electrodes in the frontal and parietal lobes of the brain, and an intracerebroventricular (ICV) guide cannula was implanted in the ventricles. The ICV guide cannula was implanted at the following coordinates: AP, −0.2 mm from bregma; ML, −1 mm; DV, −2.1 mm. After implantation, they were fixed to the skull using dental acrylic resin (Templon, GC Corporation, Tokyo, Japan). After surgery, the analgesic ibuprofen (0.4 g / 250 ml, Yung Shin Pharm. Ind. Co., Ltd.) was added to the drinking water for 7 days. Mice were allowed to recover for 7–10 days and acclimatized to routine handling. Administration of pyrogen-free saline (PFS) via intravenous vein (icv) and oral gavage was timed to coincide with the scheduled experimental administration to reduce the effects of extraneous stress prior to the experiment. Mice were housed in individual cages in a laboratory animal room with a 12:12 h cycle and a constant temperature of 23–24°C. Food and water were available ad libitum. All procedures were approved by the National Taiwan University Institutional Animal Care and Use Committee (IACUC).

[0040] EEG recording and sleep analysis EEG signals were amplified using an amplifier (Coulbourn Instruments, Lehigh Valley, PA, USA; Model V75-01). Analog band-pass filtering of 0.1 to 40 Hz was applied to the EEG, with a gain of 10,000. These filtered EEG signals were fed into an analog-to-digital converter (NI PCI-6033E, National Instruments, Austin, TX, USA) with a sample rate of 128 Hz. The digital EEG signals were saved as binary files for further sleep analysis. Sleep-wake activity was recorded and analyzed manually in 12-s epochs using custom software ICELUS (MROpp, University of Michigan) written in LabView for Windows (National Instruments). Wakefulness, NREM sleep, and REM sleep were classified according to the following criteria in rodents: Wakefulness was characterized by low-amplitude, high-frequency EEG waves. NREM sleep showed large synchronized EEG amplitudes dominated by delta waves between 0.5 and 4.0 Hz, while REM sleep showed predominant theta waves between 6.0 and 9.0 Hz. Furthermore, sleep structure, including the number of periods, period duration (minutes), and stage transitions, was also assessed.

[0041] material Three different concentrations of the Ent1 inhibitor J4 (1.0, 6.0, and 12.0 mg / kg) were dissolved in 1% 2-hydroxypropyl-beta-cyclodextin (HPβCD) solution for oral gavage administration to investigate its single-dose effect on physiological sleep. A low dose of caffeine (10 mg / kg, Sigma-Aldrich, USA) was used to induce insomnia. A1R antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, Sigma-Aldrich, USA) and A1R antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, Sigma-Aldrich, USA) were also used. 2A Both the R antagonist 5-amino-7-(β-phenylethyl)-2-(8-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261, Tocris) were dissolved in 0.5% DMSO at a dose of 5 μg / 1 μl for icv administration. PFS and 0.5% DMSO were used as vehicle controls.

[0042] Experimental procedure for testing drug concentration gradients After recovery on days 7–10 after surgery, EEG recording electrodes were implanted into the skulls of experimental mice. 30 minutes before the light-to-dark phase, mice were given 0.5 ml of PFS by oral gavage. The mice were allowed to adapt to the experimental procedure for 10 consecutive days. After oral administration of PFS on days 17–20 after surgery, 24-hour recordings were initiated for the control group. Different concentrations of the Ent1 inhibitor J4 (1.0, 6.0, or 12.0 mg / kg) were then orally administered randomly, and the same 24-hour recordings were conducted for the control group (with a 2-day interval between doses) (Figure 9). Acute insomnia model These experiments used two different methods to induce insomnia in mice. [Example]

[0043] Example 1 Stress-induced insomnia model The surgery and acclimation protocols were identical to those used in the section for testing drug concentration gradients. Next, PFS was orally administered 3 hours before the light phase on days 17–20 after surgery, followed by 24-hour recordings. In the control group, acute insomnia was induced by cage switching during the dark / light transition, and sleep fluctuations were recorded. Subsequently, different concentrations of J4 (1.0, 6.0, or 12.0 mg / kg) were orally administered randomly 48 hours later (each dose interval was 2 days), and finally, sleep changes were sequentially analyzed and compared (Figure 10).

[0044] Example 2 Caffeine-induced insomnia model The surgical and rehabilitation regimen was the same as described above. One difference from the previous experiment was that on postoperative days 17–20, the baseline group received oral PFS 3 hours before the light phase followed by 24-hour recordings. The control group received oral PFS followed by an intraperitoneal injection of caffeine 30 minutes before the light phase to simulate the sleeplessness response induced by caffeine. After 48 hours, the control group received oral administration of different concentrations of the Ent1 inhibitor J4 (1.0, 6.0, or 12 mg / kg) randomly, and the same 24-hour recordings were performed in the control group. (The dose interval was 2 days.) (Figure 11).

[0045] Immunofluorescence assay The rodents used in the experiment were housed under the same conditions as in the previous experiment. PFS was administered orally for 7 days to facilitate compliance with the experimental procedures. On the 8th day, the treatment group received an oral dose of 6 mg / kg of the Ent1 inhibitor J4 or PFS (control group). Five hours after oral delivery, the brains of the experimental mice were perfused with 4% paraformaldehyde and then excised. The experimental mice were given Zoletil 50 (8.3 mg / kg, intraperitoneally, Virbac, Carros, France) and xylazine (14.8 mg / kg, intraperitoneally, Sigma-Aldrich, USA) at 5 hours. After the mice no longer showed signs of pain, the chest was opened. To initiate PFS perfusion, a perfusion needle (also known as a butterfly needle) was placed in the left ventricle of the experimental mice. The whole blood of the experimental mice was then replaced with PFA (depending on whether the liver was bloodless) followed by the addition of 4% paraformaldehyde (P6148, Sigma-Aldrich, USA). The entire brain was quickly removed and replenished with 4% PFA (when the torso pulsation was no longer observed due to myofibril degeneration) and then impregnated with 4% PFA for 6 hours at room temperature. The entire brain was then immediately removed and incubated with 4% PFA for 6 hours at room temperature. To avoid physical damage during freezing of the sections, the entire brain was then substituted with 20% glycerol in 0.1% PB solution (Sigma-Aldrich, USA) for 24–48 hours at 4°C. The brain was then embedded in Optimal Cutting Temperature (OCT) compound (Sakura Finetek USA, Inc.) and stored at -80°C for cryosectioning. After cryoprotection, brain tissue was sectioned using the dry ice sectioning method (machine provided by Dr. Yen, College of Life Sciences, National Taiwan University) from 0.10 mm posterior to 0.14 mm anterior to bregma (the ventrolateral preoptic nucleus in adult mice). The sections were then washed three times with PBS for 5 minutes each, then incubated in PBS containing 0.3% Triton X-100 (PBST) and shaken at room temperature for 15 minutes. To reduce nonspecific binding, brain sections were blocked with 3% normal goat serum (NGS; Jackson Immunoresearch) in PBS.At the end of this step, slices were stained with the following primary antibody: anti-fos (1:1000; ab190289; Abcam) and incubated for 16 h at 4 °C. At the end of the incubation, slices were washed three times with PBS for 5 min each and then incubated for 1 h with the corresponding secondary antibody: goat anti-rabbit IgG H&L Alexa Fluor® 594 (1:500; ab150080; Abcam). Finally, slices were washed three times with PBS for 5 min each.

[0046] At the end of the above steps, to visualize the cells more easily, nuclei were stained with 100 ng / mL DAPI (D5242, Sigma-Aldrich) in PBS for 10 min. The slices were then rinsed six times with PBS for 10 min each. After air-drying to remove excess water, each brain slice was covered with a 0.17 mm thick coverslip (AP-0810401, Sigma-Aldrich) and a few drops of Fluoromount™ aqueous mounting medium (F4680, Sigma-Aldrich) were applied. Fluorescence images were taken using an Olympus IX83 inverted fluorescence microscope (provided by Dr. Pei-Hsueh Tsai, College of Veterinary Medicine, National Taiwan University).

[0047] statistical analysis All data were expressed using the mean and standard error of the mean (SEM), and results were analyzed using SPSS. Differences between groups were calculated using independent sample t-tests and one-way analysis of variance. Statistical significance was determined based on the following criteria: * :P<0.05 (significant).

[0048] result Effects of oral Ent1 inhibitor J4 on physiological sleep in experimental mice during the light phase Oral administration of three different concentrations of the Ent1 inhibitor J4 (1.0, 6.0, and 12.0 mg / kg) was compared with a control group (1% HPβCD) within 24 hours of administration. Results showed no significant changes in 12-hour NREM sleep (ZT1-12, light phase) after administration of the three different concentrations of J4 30 minutes before the light phase (Figures 12-14). On the other hand, oral gavage administration of the three different concentrations of J4 significantly increased NREM sleep (Figure 15) and decreased the proportion of wakefulness from 13 to 18 hours (ZT13-18) (Figures 16-18). Furthermore, there was no significant change in REM sleep after administration of the three different concentrations of J4 (Figures 19-21).

[0049] Effects of oral Ent1 inhibitor J4 on physiological sleep in experimental mice during the dark period The same method was used to test the effect of the Ent1 inhibitor J4 on sleep as described above. Three different concentrations of J4 (1.0, 6.0, and 12.0 mg / kg) were administered orally via gavage relative to the control (1% HPβCD) during the first 30 min of the dark phase, and sleep-wake activity was recorded for 24 h. Results showed a tendency for NREM sleep to increase from 1 to 3 h (ZT13-15) after administration of the three different concentrations of J4, but no significant differences were observed (Figure 22). On the other hand, three different doses of J4 (1.0, 6.0, and 12.0 mg / kg) significantly increased NREM sleep and decreased it from 4 to 6 h (ZT16-18) (Figure 22). Furthermore, there was no significant change in REM sleep between ZT13 and ZT18 after administration of the three different concentrations of J4 (Figures 23-25). Ent1 inhibitor J4 blocked caffeine-induced insomnia The present invention aimed to first determine whether Ent1 inhibitors, after determining the onset time of Ent1 inhibitors, exhibit a blocking effect on caffeine-induced insomnia. First, IP injection of caffeine during the dark-light period resulted in caffeine-induced insomnia. Referring to the results shown in Figures 26-28, a statistically significant decrease was observed at ZT1-2 after oral administration of caffeine compared with that after administration of 1% HPβCD and PFS. Next, we further analyzed the changes in 24-hour NREM sleep during caffeine-induced insomnia using different concentrations of the Ent1 inhibitor J4. Two doses of J4 (1 mg / kg and 12 mg / kg) significantly blocked the caffeine-induced decrease in NREM sleep (Figures 29-34) and the increase in wakefulness during ZT1-2 (Figure 35, parts A and C). Administration of 6 mg / kg of J4 only tended to block the caffeine-induced increase in NREM sleep and decrease in wakefulness, without any significant difference (Figure 35, part B). (Veh = vehicle)

[0050] Ent1 inhibitor J4 blocked stress-induced insomnia In addition to caffeine-induced insomnia, cage exchange was used to induce two stages of sleep: an early stress response and acute insomnia. First, in the group treated with oral PFS and the group treated with oral PFS and cage exchange at the dark-light transition, a statistically significant reduction in the early stress response accompanied by a reduction in NREM sleep was observed within 1 to 4 hours (ZT1-4) after cage exchange. There was no significant difference between ZT5-9 and ZT10-18. Acute insomnia symptoms persisted for 9 hours after cage exchange (Figures 36-39). Based on the results, oral administration of PFS was compared with oral administration of three different concentrations of J4 (1.0, 6.0, and 12.0 mg / kg) accompanied by cage exchange. It was found that the Ent1 inhibitor J4 blocked acute insomnia induced by cage exchange (Figures 40-49). There was also no significant difference in REM sleep.

[0051] The effect of Ent1 inhibitor J4 on sleep was mediated by adenosine receptors Previous results have demonstrated that oral administration of an Ent1 inhibitor significantly increased NREM sleep and improved acute insomnia. Furthermore, oral administration of an Ent1 inhibitor also improved caffeine-induced insomnia. Caffeine promotes the production of A1 and A2. 2A This experiment therefore demonstrated that oral gavage administration of Ent1 inhibitor J4 inhibits the A1 or A2 adenosine receptors. 2A We further demonstrated whether sleep promotion was achieved by activating type 2 adenosine receptors.

[0052] The A1R antagonist DPCPX blocked the effect of the Ent1 inhibitor J4. To exclude the effects of intravenous injection, we compared the following four treatments. These four groups were: baseline group: untreated, followed by drug administration after acclimation; vehicle (1% HPβCD) + DMSO group: oral administration of 1% HPβCD followed by intravenous injection of DMSO 3.5 hours after drug administration (ZT15); Ent1 inhibitor J4 + DMSO group: oral administration of J4 followed by intravenous injection of DMSO 3.5 hours after drug administration (ZT15); and Ent1 inhibitor J4 + DPCPX group: oral administration of J4 followed by intravenous injection of DPCPX (adenosine A1 receptor antagonist) 3.5 hours after drug administration (ZT15). Results revealed no significant changes in NREM sleep in the baseline and vehicle + DMSO groups during the 24-hour recording period (Figures 50–52). Consistent with previous results, J4 also increased NREM sleep and decreased wakefulness, but not REM sleep, in these groups (Figures 53-55). Application of the A1R antagonist DPCPX significantly blocked the increase in NREM sleep and decrease in wakefulness induced by the Ent1 inhibitor (Figures 56 and 57).

[0053] A 2A The R antagonist SCH58261 blocked the effect of the Ent1 inhibitor J4. In addition to the above, the effect of SCH58261 (adenosine 2A receptor antagonist) on the Ent1 inhibitor J4 was investigated. J4 was orally administered, and SCH58261 was injected intravenously 3 hours after administration (ZT15). The results showed that A 2A Application of the R antagonist SCH58261 also significantly blocked the increase in NREM sleep and the decrease in wakefulness induced by the Ent1 inhibitor, but not REM sleep. However, the decrease was not as large as in the icv-injected DPCPX group, and there was still no significant difference in REM sleep (Figure 58).

[0054] Consideration Efficacy of Ent1 inhibitor J4 on sleep The study was conducted using oral administration of the substance and undisturbed experimental mice. The Ent1 inhibitor J4 was administered at three concentrations (1.0, 6.0, and 12 mg / kg) 30 minutes before the mice were about to enter the active or resting phase. During the dark phase, NREM sleep was significantly increased for 4 to 6 hours after administration of the three doses of the Ent1 inhibitor J4, while during the light phase, NREM sleep was significantly increased for 13 to 18 hours after administration. In summary, after oral administration, J4 not only induced sleepiness during the dark phase (active phase), but also increased and prolonged NREM sleep during the light phase (sleep phase).

[0055] Ent1 inhibitor J4 improved insomnia In this study, we used two insomnia models: acute stress-induced insomnia and caffeine-induced insomnia. These two models were chosen because insomnia is known to be caused by a variety of factors, including stress, chronic pain, lifestyle, excessive excitement, and drugs such as caffeine. As a nonselective adenosine antagonist, caffeine is present in daily life. While most people consume caffeine as a cooling agent, inappropriate caffeine use can lead to varying degrees of sleep disruption. In many sleep-related studies, caffeine has been used to demonstrate transient insomnia. In this study, administration of the Ent1 inhibitor J4 reduced and blocked the pharmacological properties of caffeine. Results indicated that J4 could reduce the pharmacological properties of caffeine.

[0056] Ent1 inhibitor J4 as a potential hypnotic agent Although absorption efficiency differs between humans and mice, the above results suggest that taking the Ent1 inhibitor J4 3 hours before bedtime can promote stable nighttime sleep. Currently, there are two main types of sleep medications on the market: benzodiazepines (BZDs) and non-benzodiazepines (non-BZDs). Major side effects include addiction and withdrawal symptoms, which, in addition to their sleep-inducing effects, also cause anxiolytic effects, muscle relaxation, and memory impairment. Furthermore, many drugs may cause dependence / abuse-related problems, muscle relaxation, and memory impairment, which may be strongly associated with falls in elderly patients. The effects of J4 alone on sleep are likely superior to those of BZDs and similar to those of dual orexin receptor antagonists (DORAs), a new class of sleep medications recently released on the market. Both drugs bind to two G protein-coupled receptors (orexin receptors-1 and -2). In addition to DORA, the typical aminobutyric acid receptor agonist sedative-hypnotic treatment for insomnia may offer an additional alternative pharmaceutical strategy by targeting the adenosine receptor system. Meanwhile, this new compound, J4, affects A1R, which is distributed throughout the brain, and can indirectly regulate the performance of wakefulness neurons. Furthermore, it also affects A1R, a GABAergic neuron that is responsible for directly increasing NREM sleep.2A It may also affect R. Therefore, J4 may have potential as a hypnotic agent.

[0057] The many features and advantages of the present disclosure are apparent from the detailed specification, and it is, therefore, intended by the appended claims to cover all such features and advantages of the present disclosure that fall within the true spirit and scope of the present disclosure. Further, because numerous modifications and variations will readily occur to those skilled in the art, it is not desired to limit the disclosure to the exact construction and operation shown and described, and therefore, all suitable modifications and equivalents may be utilized that fall within the scope of the present disclosure.

[0058] Moreover, those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be utilized as a basis for designing other structures, methods and systems for carrying out the several purposes of the present disclosure. Accordingly, the scope of the appended claims should not be deemed limited by the foregoing description.

Claims

1. Compounds of Formula (I), (II) or (III) 【Chemistry 1】 A method of treating a sleep disorder in a subject in need thereof, comprising administering to the subject a pharmaceutically acceptable salt thereof, or a composition thereof, wherein X is a halogen.

2. 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.

3. 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.

4. 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.

5. The 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 6 -[(5-chlorothien-3-yl)methyl]adenosine.

6. 10. The method of claim 1, wherein the therapeutically effective amount of the compound is 0.5 to 15 mg / kg.

7. 10. The method of claim 1, wherein the compound, a pharmaceutically acceptable salt thereof, or a composition thereof is administered by oral, intranasal, intravenous, intramuscular, subcutaneous, intraperitoneal, or topical route.

8. The method of claim 1 , wherein the composition further comprises a pharmaceutically acceptable carrier, excipient, or vehicle.

9. 10. The method of claim 1, wherein the subject has insomnia or Alzheimer's disease (AD).

10. 10. The method of claim 9, wherein the insomnia is stress-induced insomnia, caffeine-induced insomnia, or a combination thereof.

11. Compounds of Formula (I), (II) or (III) 【Chemistry 2】 or a pharmaceutically acceptable salt thereof, wherein X is a halogen.

12. 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.

13. 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.

14. 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.

15. The 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 6 -[(5-chlorothien-3-yl)methyl]adenosine.

16. The composition for use according to claim 11, wherein the therapeutically effective amount of the compound is 0.5 to 15 mg / kg.

17. 12. The composition for use according to claim 11, wherein the composition is administered by oral, nasal, intravenous, intramuscular, subcutaneous, intraperitoneal or topical route.

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

19. 12. The composition for use according to claim 11, wherein the subject has insomnia or AD.

20. 20. The composition of claim 19, wherein the insomnia is stress-induced insomnia, caffeine-induced insomnia, or a combination thereof.

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