WO2024145562A1 - Method of treating sleep disruptions and composition for use therein - Google Patents
Method of treating sleep disruptions and composition for use therein Download PDFInfo
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- WO2024145562A1 WO2024145562A1 PCT/US2023/086376 US2023086376W WO2024145562A1 WO 2024145562 A1 WO2024145562 A1 WO 2024145562A1 US 2023086376 W US2023086376 W US 2023086376W WO 2024145562 A1 WO2024145562 A1 WO 2024145562A1
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- adenosine
- methyl
- sleep
- entl
- inhibitor
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- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
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- A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
Definitions
- the present disclosure relates to a method of treating a sleep disruption and a composition for use in a method of treating a sleep disruption.
- Insomnia is one of the most common sleep problems, which is believed to affect 30 to 40% of the general population. It is a sleeping problem that makes hard to get to sleep. Poor sleep quality, the inability to maintain a good sleep (waking), difficulty falling asleep, or — most frequently among teenagers — the sensation of not being fully and soundly asleep despite having slept for an extended period. Long-term insomnia lasts longer than a month, while short-term insomnia lasts a few days or weeks. Psychological stress, chronic pain, certain drugs, lifestyle variables, caffeine, alcohol, and stress are the most prevalent causes of insomnia.
- the positively charged microtubule-binding domain of tau When the positively charged microtubule-binding domain of tau is affected by hyperphosphorylation, it loses its positively charged and dissociates from the microtubules. Dissociated tau protein further aggregates into paired helical filaments that ultimately forms the insoluble neurofibrillary tangle (NFT) in the axons.
- NFT neurofibrillary tangle
- Ap plaques and NFT are toxic to neurons that interfere with the synaptic transmission and cellular function, and eventually lead to neuronal apoptosis.
- the neurotoxic A ⁇ plaques and tau tangles cause oxidative stress, neuroinflammation, mitochondria dysfunction and DNA damage, which lead to the symptoms of AD.
- the sleep disruption has been documented as a prevalent symptom of AD, and this issue has a serious influence on patients and/or caregivers.
- the sleep disruption like hard to fall asleep, sleep fragmentation, disturbance in circadian rhythm, sleep in the daytime and arousal at night.
- Researches focusing on the relationship between sleep and pathological markers depict that the degree of cortical A ⁇ measured by the PET scanning is correlated with the decrement of non-rapid eye movement (NREM) sleep and the downturn of NREM sleep is associated with tauopathy in the early AD patients.
- NREM non-rapid eye movement
- Ap and p-tau are also the key factors on the sleep disturbance in the AD. Based on the several studies have speculated, sleep disorder is directly associated with cognitive impairment.
- the present invention provides a new method of treating a sleep disruption by using the compounds of adenosine analogues.
- a method of treating a sleep disruption in a subject in need thereof including administering to the subject a compound of formula (I), (II) or (III):
- compositions for use in a method of treating a sleep disruption in a subject in need thereof including administering to the subject the composition including a compound of formula (I), (II) or (III) as shown above.
- the compound is selected from the group consisting of 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.
- the compound is selected from the group consisting of N 6 -[(5-iodothien-2-y[)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.
- 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 -y l)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, and
- a therapeutically effective amount of the compound is 0.5-15 mg/kg, preferably 1-12 mg/kg.
- the compound, a pharmaceutically acceptable salt thereof, or a composition thereof is administered by an oral, nasal, intravenous, intramuscular, subcutaneous, intraperitoneal or topical route.
- the composition further includes a pharmaceutically acceptable carrier, excipient or vehicle.
- the subject has insomnia or Alzheimer’s disease (AD).
- AD Alzheimer’s disease
- the insomnia is stress-induced insomnia, caffeine-induced insomnia or a combination thereof.
- the present invention at least provides the following advantages:
- the present invention can exhibit benefits on the regulation of disrupted homeostatic sleep induced by the AD, stress and/or caffeine.
- the present invention may alleviate the symptoms including sleep disruption of AD so as to slow down the deterioration rate thereof.
- FIGs. 1A and IB are stained images showing Ap plaques distributed in the CAI, CA3 and hilus of the hippocampus of the control mice and the mice receiving icv-STZ and ih-A ⁇ , respectively.
- FIG. 1C illustrates the ratio of Ap positive area/tissue area of the mice receiving icv- STZ and ih-A ⁇ in comparison with the control mice.
- FIGs. 1D-1F illustrate the levels of phosphorylated tau protein at the residue Ser404 in the hippocampus of the mice receiving icv-STZ and ih-A ⁇ in comparison with the control mice.
- FIG. 2 A and 2B are stained images showing the number of ChAT-positive neurons in the MSDB of the control mice and the mice receiving icv-STZ and ih-A ⁇ , respectively, according to an embodiment of the present invention.
- FIG. 2C is a stained image showing the number of ChAT-positive neurons in the MSDB of the control mice treated with HP ⁇ CD according to an embodiment of the present invention.
- FIG. 2D is a stained image showing the number of ChAT-positive neurons in the
- MSDB of the mice receiving icv-STZ and ih-A ⁇ treated with the Entl inhibitor J4 according to an embodiment of the present invention MSDB of the mice receiving icv-STZ and ih-A ⁇ treated with the Entl inhibitor J4 according to an embodiment of the present invention.
- FIG. 2E is a histogram related to the statistical result of FIGs. 2A-2D according to an embodiment of the present invention.
- FIG. 3 illustrates the result of a NOR test according to an embodiment of the present invention.
- FIG. 4A illustrates the result of escape latency in a MWM test according to an embodiment of the present invention.
- FIG. 4B illustrates the heat maps of the probe test in MWM according to an embodiment of the present invention.
- FIG. 4C illustrates the statistical result of the probe test in MWM according to an embodiment of the present invention.
- FIG. 5 illustrates the effect of the Entl inhibitor J4 on the expression of NO in the sAD mice according to an embodiment of the present invention.
- FIG. 6A illustrates the effect of the Entl inhibitor J4 on DNA damage in the sAD mice according to an embodiment of the present invention.
- FIG. 6C illustrates the effect of the Entl inhibitor J4 on the activity of DNA-PKcs in the sAD mice according to an embodiment of the present invention.
- FIGs. 7A-7C illustrate time-courses of NREM sleep. REM sleep and wakefulness in the sAD mice, respectively, according to an embodiment of the present invention.
- FIGs. 7D-7F illustrate time-courses of NREM sleep, REM sleep and wakefulness with the Entl inhibitor J4 treatment in the sAD mice, respectively, according to an embodiment of the present invention.
- FIGs. 8A and 8B illustrate the summary of each group on NREM sleep during 1 -3 hrs in the dark period and 15-17 hrs in the light period according to an embodiment of the present invention.
- FIG. 8C illustrates the summary of each group on REM sleep during 13-23 hrs in the light period according to an embodiment of the present invention.
- FIGs. 8D and 8E illustrate the summary of each group on wakefulness during 1-3 hrs in the dark period and 15-21 hrs in the light period according to an embodiment of the present invention.
- FIG. 8F-8I illustrate bout number and duration of NREM and REM according to an embodiment of the present invention.
- FIG. 8 J illustrates the number of transitions in each group in the light period according to an embodiment of the present invention.
- FIG. 10 illustrates the experimental procedure for acute insomniac mice according to an embodiment of the present invention.
- FIG. 12 illustrates the analysis of 24-h NREM sleep alterations after vehicle control (1% HP ⁇ CD) and Entl inhibitor J4 at 1.0 mg/kg. *: indicates a statistically significant difference (p ⁇ 0.05) compared to the control group (orally administered 1% HP ⁇ CD).
- FIG. 23 illustrates the analysis of 24-h REM sleep after oral administering 1.0 mg/kg Entl inhibitor J4 and vehicle control (1 % HP ⁇ CD).
- FIG. 34 illustrates Entl inhibitor J4 (12 mg/kg) blocked caffeine-induced increase of wakefulness. *: indicates a statistically significant difference (p ⁇ 0.05) compared to the control group (orally administered PFS).
- FIG. 42 illustrates oral administration of 12 mg/kg Entl inhibitor J4 blocked cage exchange-induced acute insomnia (decreases of NREM sleep). *: indicates a statistically significant difference (p ⁇ 0.05) compared to the control group (orally administered PFS).
- FIG. 52 illustrates the effects of vehicle and 0.5 % DMSO on wakefulness.
- FIG. 54 illustrates the effect of Entl inhibitor J4 (6 mg/kg) on REM sleep. *: indicates a statistically significant difference (p ⁇ 0.05) compared to the control group (baseline).
- FIG. 58 illustrates SCH58261 blocked Entl inhibitor J4-induced increases of NREM sleep.
- the dose of oral Entl inhibitor J4 was 6 mg/kg and HP ⁇ CD was 1%; icv injection DMSO was 5% and SCH58261 was 5 pg/lpl. Data are expressed as mean ⁇ SEM. vehicle control versus Entl inhibitor J4 + DMSO, #p ⁇ 0.05; Entl inhibitor J4 + DMSO versus Entl inhibitor + SCH58261, &p ⁇ 0.05.
- This behavioral task was talcing place in a white acrylic open box (40x40x45 cm) and divided into three phases: habituation, training and testing.
- the mice were allowed to freely explore the apparatus for 15 min (used as OFT) to become familiar with the surroundings. No objects were placed in the box during the habituation phase. Twenty-four hours later, the two identical objects (Al and A2) were arranged in a diagonal position and were allowed to explore for 10 min.
- mice were placed back into the home cage, rested within the inter trial interval (90 min) and prepared for the testing phase.
- the task was conducted in a circular pool (153 cm in diameter) filled with water (25 ⁇ 1°C), which mixed with non-toxic white paint and turned transparent water to opaque, and the pool was divided into four quadrants.
- An escape platform (10 cm in diameter) was placed into quadrant 4 (target quadrant) and submerged 2 cm below the surface of water.
- the visual cues were pasted on the edge of the pool to provide the orientation for mice.
- all the mice were given three trials per session for five consecutive days (days 19, 20, 21, 22, 23), and allowed them to search the hidden platform for 120 s in each trial. After they climbed on the platform, the mice had left on it for 30 s before returning to the home cage.
- mice failed to find the platform, they would be gently guided and allowed to stay on platform for 30 s.
- the probe test (on day 24) was given by removing the escape platform from the target quadrant, and the mice were allowed to freely swim for 120 s.
- the escape latency meaning the time taken to reach the platform in the training phase was evaluated. Time spent in the target quadrant and the number of times passed through the original position of the platform in the probe test were measured by video tracking software (EthoVision XT version 14.0.1322, Noldus Information Technology by the Netherlands).
- mice treated with icv-STZ and ih-A ⁇ exhibited 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) when compared with the neuron numbers of 60.11 ⁇ 2.98 obtained from the control group (FIGs. 2A, 2B, 2E).
- the sAD mice treated with Entl inhibitor J4 elucidated that the cholinergic neuronal loss could be prevented, and the number of cholinergic neurons in MSDB maintained as 51.11 ⁇ 1.15 (vs.
- mice exhibited the preference to stay in the target quadrant (quadrant II), where the hidden platform existed during the training sessions, in the probe test when the hidden platform was removed.
- Mice treated with HP ⁇ CD did not change the preference for mice to stay in quadrant II in the probe test.
- Mice treated with ih-A ⁇ and icv-STZ lost the preference to stay in quadrant II, indicating the impairment of spatial memory'; while J4 could reverse the memory impairment in the sAD mice.
- Wakefulness was markedly decreased from 88.31 ⁇ 4.24 % to 59.02 ⁇ 2.9 % (p ⁇ 0.05, one-way ANOVA with post hoc comparison) during ZT13-15 (FIG. 8D) and significantly increased from 23.81 ⁇ 3.11 % to 46.68 ⁇ 2.81 % (p ⁇ 0.05, one-way ANOVA with post hoc comparison) during ZT’3-9 in the sAD mice (FIGs. 7C and 8E).
- FIGs. 7E and 8C A ⁇ ⁇ STZ, one-way ANOVA with post hoc comparison, FIGs. 7E and 8C). J4 also respectively reversed the decreased wakefulness during ZT13-15 and enhancement of wakefulness during ZT’3-9 back to 82.12 ⁇ 4. 16 % (p ⁇ 0.05 vs. A ⁇ ⁇ STZ, one-way ANOVA with post hoc comparison, FIGs. 7F and 8D) and 28.38 ⁇ 3.0 % (p ⁇ 0.05 vs. A ⁇ + STZ, one- way ANOVA with post hoc comparison, FIGs. 7F and 8E).
- the results of the present invention indicated that treatment with the Entl inhibitor J4 normalized the levels of nitric oxide, cleaved-caspase 3 and phosphorylated H2A histone family member X (y-H2AX), and increased activities of nuclear DNA-dependent serine/threonine protein kinase (DNA-PKcs) through the non-homologous end joining (NHEJ) pathway to repair double-strand breaks in DNA.
- J4 also alleviated the loss of cholinergic neurons in the medial septum-diagonal band of Broca, and further improved cognitive deficits.
- the sAD mice increased NREM sleep during dark period and decreased NREM and REM sleep during light period, while the elevation of extracellular adenosine exhibited beneficial for the homeostatic sleep.
- treatment with the Entl inhibitor in the present invention normalized the disrupted sleep pattern in the sAD mice, suggesting adenosine augmentation evoked by the Entl inhibitor has positive effects on regulating homeostatic sleep.
- Adenosine is a sleep promoting substance, and blocking the Entl results in the increased extracellular adenosine. Therefore, it can be assumed that it would enhance the binding between the adenosine receptor and adenosine, which further regulates sleep in the CNS.
- the Entl inhibitor J4 may be potential for sAD treatment.
- mice were hosted in the individual cage with the constant temperature at 23-24°C in the 12:12h cycle laboratory animal room. All food and water were provided ad libitum. All these procedures were approved by the National Taiwan University Institutional Animal Care and Use Committee (IACUC).
- IACUC Institutional Animal Care and Use Committee
- the vigilances of wakefulness, NREM sleep, and REM sleep were categorized according to the following criteria in rodents.
- wakefulness low amplitude and high-frequency EEG waves were presented.
- a synchronized large-EEG amplitude dominating with 0.5-4.0 Hz delta waves were shown during NREM sleep, and a dominant 6.0-9.0 Hz theta waves were displayed in REM sleep.
- the sleep architectures including bout numbers, bout duration (min) and stages transitions, were also assessed.
- Entl inhibitor J4 1.0, 6.0, 12.0 mg/kg
- HP ⁇ CD 2-Hydroxypropyl-beta-cyclodextin
- the nuclei were stained with DAPI (D5242, Sigma-Aldrich) in PBS solution at a concentration of 100 ng/mL for 10 minutes, and the slices were rinsed six times with PBS for 10 minutes each. After airdrying to remove excess water, each brain slice was covered with a 0.17 mm thick coverslip (AP-0810401, Sigma- Aldrich) and few drops of fluoromountTM aqueous mounting media (F4680, Sigma-Aldrich). The Olympus 1X83 inverted fluorescence microscope was used to capture the fluorescent images (provided by Dr. Pei-Hsueh Tsai, Department of Veterinary Medicine, National Taiwan University).
- SCH58261 (adenosine 2A receptor antagonist) on Entl inhibitor J4 was performed. J4 was administered orally with icv injection SCH58261 three hours after administration (ZT15). The results showed that application of AZAR antagonist SCH58261 also significantly blocked Entl inhibitor induced increases of NREM sleep and the decreases of wakefulness, but not REM sleep. However, the decrease was not as great as in the icv injection DPCPX group, and there was still no significant difference in REM-Sleep (FIG. 58).
- Entl inhibitor J4 was administered in three concentration gradients (1.0, 6.0, 12 mg/kg) 30 minutes before the mice were about to enter the active or resting phase. In the dark phase, there was a significant increase in NREM sleep for 4-6 hours after three doses of Entl inhibitor J4, while in the light phase there was a significant increase in NREM sleep for 13-18 hours after given. Taken together, J4 not only caused drowsiness in the dark phase (active phase), but also increased and prolonged NREM sleep in the light phase (sleep phase) after oral administration.
- Entl inhibitor J4 can be taken 3 hours before bedtime to help stabilize sleep during the night.
- BZD benzodiazepines
- non-BZD non-benzodiazepines
- the major adverse side effects include addiction and withdrawal symptoms, which cause anxiolytic effects, muscle relaxation, and memory impairment in addition to sleep-inducing effects. More often, it may cause dependency/ abuse related problems, and muscle relaxation and memory impairment, and may be strongly associated with falls in the elderly.
- J4 on sleep alone is probably better than that of BZD drugs, and is similar to that of Dual Orexin Receptor Antagonists (DORAs), a new type of sleeping drug that has just hit the market, both combining with two G protein couple receptors (orexin receptor- 1 and -2).
- DORAs Dual Orexin Receptor Antagonists
- the typical aminobutyric acid receptor agonist sedative hypnosis treatment for insomnia may offer an extra and alternative pharmaceutical strategy by targeting the adenosine receptor system.
- this new compound can affect the AiR, which is distributed throughout the brain, indirectly regulating the performance of the waking neuron, and furthermore, it can also affect the A 2 AR, the GABAergic neuron responsible for directly increasing NREM sleep.
- J4 may be potential as hypnotic drug.
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Abstract
A method of treating a sleep disruption in a subject in need thereof is provided, including administrating to the subject a compound of formula (I), (II) or (III) or a pharmaceutically acceptable salt thereof, wherein X is halogen. A composition for use in a method of treating a sleep disruption in a subject in need thereof, including administering to the subject in need thereof the composition comprising a compound of formula (I), (II) or (III) as shown above is also provided.
Description
METHOD OF TREATING SLEEP DISRUPTIONS AND COMPOSITION FOR USE THEREIN
FIELD OF INVENTION
[0001] The present disclosure relates to a method of treating a sleep disruption and a composition for use in a method of treating a sleep disruption.
BACKGROUND OF THE INVENTION:
[0002] Insomnia is one of the most common sleep problems, which is believed to affect 30 to 40% of the general population. It is a sleeping problem that makes hard to get to sleep. Poor sleep quality, the inability to maintain a good sleep (waking), difficulty falling asleep, or — most frequently among teenagers — the sensation of not being fully and soundly asleep despite having slept for an extended period. Long-term insomnia lasts longer than a month, while short-term insomnia lasts a few days or weeks. Psychological stress, chronic pain, certain drugs, lifestyle variables, caffeine, alcohol, and stress are the most prevalent causes of insomnia.
[0003] Over the past decades, Alzheimer’s disease (AD) is the most common chronic neurodegenerative disorder in the elderly and accounts for 50 million people suffering from the disease. The typical pathological hallmarks for diagnosis of AD are the progressively extracellular aggregation of amyloid-beta (Aβ) plaques and phosphorylated-tau (p-tau) accumulation in brains after postmortem examination. The excessive neurotoxic Aβ undergoes conformational changes, oligomerization and aggregation, and subsequently forms plaques and disrupts the physiology functions of the brain. 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 positively charged and dissociates from the microtubules. Dissociated tau protein further aggregates into paired helical filaments that ultimately forms the insoluble neurofibrillary tangle (NFT) in the axons. Ap plaques and NFT are toxic to neurons that interfere with the synaptic transmission and cellular function, and eventually lead
to neuronal apoptosis. The neurotoxic Aβ plaques and tau tangles cause oxidative stress, neuroinflammation, mitochondria dysfunction and DNA damage, which lead to the symptoms of AD.
[0004] Besides the cognitive deficits, the sleep disruption has been documented as a prevalent symptom of AD, and this issue has a serious influence on patients and/or caregivers. The sleep disruption like hard to fall asleep, sleep fragmentation, disturbance in circadian rhythm, sleep in the daytime and arousal at night. Researches focusing on the relationship between sleep and pathological markers depict that the degree of cortical Aβ measured by the PET scanning is correlated with the decrement of non-rapid eye movement (NREM) sleep and the downturn of NREM sleep is associated with tauopathy in the early AD patients. In general, Ap and p-tau are also the key factors on the sleep disturbance in the AD. Based on the several studies have speculated, sleep disorder is directly associated with cognitive impairment. Patients with mild cognitive impairment (MCI) and AD show a 40 % decrease of K-complexes density during sleep, which is a cause for cognitive deficits. Furthermore, according to the questionnaire survey, 24.5 % of mild to moderate AD patients suffer from sleep disorders, and the ratio may getting higher in next three decades. Therefore, sleep disturbance is a possible risk factor that can further accelerate the disease progression of AD. Nevertheless, there is none of medication successfully relieving the symptoms of AD.
SUMMARY OF THE INVENTION
[0005] Based on the above reasons, the present invention provides a new method of treating a sleep disruption by using the compounds of adenosine analogues.
[0006] In an aspect of the present invention, a method of treating a sleep disruption in a subject in need thereof, including administering to the subject a compound of formula (I), (II) or (III):
[0007] In another aspect of the present invention, a composition for use in a method of treating a sleep disruption in a subject in need thereof, including administering to the subject the composition including a compound of formula (I), (II) or (III) as shown above.
[0008] Preferably, the compound is selected from the group consisting of N6 -[(3-halothien-2- yl)methyl] adenosine, N6 -[(4-halothien-2-yl)methyl]adenosine, and N6-[(5-halothien-2- yl)methyl] adenosine. More preferably, the compound is selected from the group consisting of N6-[(5-iodothien-2-y[)methyl]adenosine, N6 -[(4-iodothien-2-yl)methyl]adenosine, N6 -[(3- iodothien-2-yl)methyl]adenosine, N6 -[(5-bromothien-2-yl)methyl]adenosine, N6 -[(4- bromothien-2-yl)methyl]adenosine, N6 - [(3 -bromothien-2-yl)methyl] adenosine. N6 - [(5 - chlorothien-2-yl)methyl]adenosine, N6 - [(4-chlorothien-2-yl)methyl] adenosine, and N6 -[(3- chlorothien-2 -yl)methyl] adenosine.
[0009] Preferably, the compound is selected from the group consisting of N6 -[(2-halothien-3- yl)methyl] adenosine, N6- [(4-halothien-3-yl)methyl] adenosine, and N6 -[(5-halothien-3- yl)methyl] adenosine. More preferably, the compound is selected from the group consisting of N6- [(2 -iodothien-3-yl)methyl] adenosine, N6 -[(4-iodothien-3-yl)methyl]adenosine, N6 -[(5- iodothien-3-yl) [methyl] adenosine, N6 - [(2-bromothien-3 -y l)methyl] adenosine, N6 - [(4- bromothien-3-yl)methyl]adenosine, N6 -[(5-bromothien-3-yl)methyl]adenosine N6 -[(2- chlorothien-3-yl)methyl]adenosine, N6 -[(4-chlorothien-3-yl)methyl]adenosine, and N6 -[(5- chlorothien-3 -yl)methyl] adenosine.
[0010] Preferably, a therapeutically effective amount of the compound is 0.5-15 mg/kg, preferably 1-12 mg/kg.
[0011] Preferably, the compound, a pharmaceutically acceptable salt thereof, or a composition thereof is administered by an oral, nasal, intravenous, intramuscular, subcutaneous, intraperitoneal or topical route.
[0012] Preferably, the composition further includes a pharmaceutically acceptable carrier, excipient or vehicle.
[0013] Preferably, the subject has insomnia or Alzheimer’s disease (AD).
[0014] Preferably, the insomnia is stress-induced insomnia, caffeine-induced insomnia or a combination thereof.
[0015] Therefore, the present invention at least provides the following advantages:
1. The present invention can exhibit benefits on the regulation of disrupted homeostatic sleep induced by the AD, stress and/or caffeine.
2. The present invention may alleviate the symptoms including sleep disruption of AD so as to slow down the deterioration rate thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIGs. 1A and IB are stained images showing Ap plaques distributed in the CAI, CA3 and hilus of the hippocampus of the control mice and the mice receiving icv-STZ and ih-Aβ, respectively.
[0017] FIG. 1C illustrates the ratio of Ap positive area/tissue area of the mice receiving icv- STZ and ih-Aβ in comparison with the control mice.
[0018] FIGs. 1D-1F illustrate the levels of phosphorylated tau protein at the residue Ser404 in the hippocampus of the mice receiving icv-STZ and ih-Aβ in comparison with the control mice. [0019] FIG. 2 A and 2B are stained images showing the number of ChAT-positive neurons in the MSDB of the control mice and the mice receiving icv-STZ and ih-Aβ, respectively, according to an embodiment of the present invention.
[0020] FIG. 2C is a stained image showing the number of ChAT-positive neurons in the MSDB of the control mice treated with HPβCD according to an embodiment of the present invention.
[0021] FIG. 2D is a stained image showing the number of ChAT-positive neurons in the
MSDB of the mice receiving icv-STZ and ih-Aβ treated with the Entl inhibitor J4 according to an embodiment of the present invention.
[0022] FIG. 2E is a histogram related to the statistical result of FIGs. 2A-2D according to an embodiment of the present invention.
[0023] FIG. 3 illustrates the result of a NOR test according to an embodiment of the present invention.
[0024] FIG. 4A illustrates the result of escape latency in a MWM test according to an embodiment of the present invention.
[0025] FIG. 4B illustrates the heat maps of the probe test in MWM according to an embodiment of the present invention.
[0026] FIG. 4C illustrates the statistical result of the probe test in MWM according to an embodiment of the present invention.
[0027] FIG. 5 illustrates the effect of the Entl inhibitor J4 on the expression of NO in the sAD mice according to an embodiment of the present invention.
[0028] FIG. 6A illustrates the effect of the Entl inhibitor J4 on DNA damage in the sAD mice according to an embodiment of the present invention.
[0029] FIG. 6B illustrates the effect of the Entl inhibitor J4 on apoptosis in the sAD mice according to an embodiment of the present invention.
[0030] FIG. 6C illustrates the effect of the Entl inhibitor J4 on the activity of DNA-PKcs in the sAD mice according to an embodiment of the present invention.
[0031] FIGs. 7A-7C illustrate time-courses of NREM sleep. REM sleep and wakefulness in the sAD mice, respectively, according to an embodiment of the present invention.
[0032] FIGs. 7D-7F illustrate time-courses of NREM sleep, REM sleep and wakefulness with the Entl inhibitor J4 treatment in the sAD mice, respectively, according to an embodiment of the present invention.
[0033] FIGs. 8A and 8B illustrate the summary of each group on NREM sleep during 1 -3 hrs in the dark period and 15-17 hrs in the light period according to an embodiment of the present invention.
[0034] FIG. 8C illustrates the summary of each group on REM sleep during 13-23 hrs in the light period according to an embodiment of the present invention.
[0035] FIGs. 8D and 8E illustrate the summary of each group on wakefulness during 1-3 hrs in the dark period and 15-21 hrs in the light period according to an embodiment of the present invention.
[0036] FIG. 8F-8I illustrate bout number and duration of NREM and REM according to an embodiment of the present invention.
[0037] FIG. 8 J illustrates the number of transitions in each group in the light period according to an embodiment of the present invention.
[0038] FIG. 9 illustrates the oral administration schedule of Ent 1 inhibitor J4 according to an embodiment of the present invention.
[0039] FIG. 10 illustrates the experimental procedure for acute insomniac mice according to an embodiment of the present invention.
[0040] FIG. 11 illustrates the experimental protocol for the caffeine-induced insomniac mice according to an embodiment of the present invention.
[0041] FIG. 12 illustrates the analysis of 24-h NREM sleep alterations after vehicle control (1% HPβCD) and Entl inhibitor J4 at 1.0 mg/kg. *: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered 1% HPβCD).
[0042] FIG. 13 illustrates the analysis of 24-h NREM sleep alterations after vehicle control (1% HPβCD) and oral Entl inhibitor J4 at 6.0 mg/kg.
indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered 1% HPβCD).
[0043] FIG. 14 illustrates the analysis of 24-h NREM sleep alterations after vehicle control (1% HPβCD) and oral Entl inhibitor J4 at 12.0 mg/kg. &: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered 1% HPβCD).
[0044] FIG. 15 illustrates the alterations of NREM sleep during the ZT 13-18 hours after the administration of different concentrations of Entl inhibitor J4. The oral doses of Entl inhibitor J4 were (A) 1 mg/kg; (B) 6 mg/kg; and (C) 12 mg/kg, respectively. The data are expressed as mean ± SEM. 1 mg/kg Entl inhibitor J4 versus control, 'p < 0.05; 6 mg/kg Entl inhibitor J4 versus control, "p < 0.05; 12 mg/kg Entl inhibitor J4 versus control, &p < 0.05.
[0045] FIG. 16 illustrates the analysis of 24-h wakefulness after vehicle control (1% HPβCD) and oral Entl inhibitor J4 at 1.0 mg/kg. *: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered 1% HPβCD).
[0046] FIG. 17 illustrates the analysis of 24-h wakefulness after vehicle control (1% HPβCD) and oral Entl inhibitor J4 at 6.0 mg/kg. #: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered 1% HPβCD).
[0047] FIG. 18 illustrates the analysis of 24-h wakefulness after vehicle control (1% HPβCD) and oral Entl inhibitor J4 at 12.0 mg/kg. &: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered 1% HPβCD).
[0048] FIG. 19 illustrates the analysis of 24-h REM sleep alterations after vehicle control (1% HPβCD) and oral Entl inhibitor J4 at 1.0 mg/kg.
[0049] FIG. 20 illustrates the analysis of 24-h REM sleep alterations after vehicle control (1% HPβCD) and oral Entl inhibitor J4 at 6.0 mg/kg.
[0050] FIG. 21 illustrates the analysis of 24-h REM sleep alterations after vehicle control (1% HPβCD) and oral Entl inhibitor J4 at 12.0 mg/kg.
[0051] FIG. 22 illustrates the alterations in NREM sleep during ZT 13-15 (A) and ZT 16—18 (B) hours following administration of Entl inhibitor J4. Oral Entl inhibitor J4 dosages of 1 mg/kg, 6 mg/kg, and 12 mg/kg were randomly administered. The data are expressed as mean ± SEM. 1 mg/kg Entl inhibitor J4 versus control, *p < 0.05; 6 mg/kg Entl inhibitor J4 versus control, #p < 0.05; 12 mg/kg Entl inhibitor J4 versus control, &p < 0.05. N.S. = no significant differences.
[0052] FIG. 23 illustrates the analysis of 24-h REM sleep after oral administering 1.0 mg/kg Entl inhibitor J4 and vehicle control (1 % HPβCD).
[0053] FIG. 24 illustrates the analysis of 24-h REM sleep after oral administering 6.0 mg/kg Entl inhibitor J4 and vehicle control (1 % HPβCD).
[0054] FIG. 25 illustrates the analysis of 24-h REM sleep after oral administering 12 mg/kg Entl inhibitor J4 and vehicle control (1 % HPβCD).
[0055] FIG. 26 illustrates the alteration of NREM sleep after administration of caffeine. *: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered 1% HPβCD).
[0056] FIG. 27 illustrates the alteration of wakefulness after administration of caffeine. *: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered 1% HPβCD).
[0057] FIG. 28 illustrates the alterations in NREM sleep during ZT 1-2 (A) and in wakefulness ZT 1-2 (B) hours following caffeine administration. *: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered PFS).
[0058] FIG. 29 illustrates Entl inhibitor J4 (1.0 mg/kg) blocked caffeine-induced insomnia. *: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered PFS).
[0059] FIG. 30 illustrates Entl inhibitor J4 (6.0 mg/kg) blocked caffeine-induced insomnia. *: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered PFS).
[0060] FIG. 31 illustrates Entl inhibitor J4 (12 mg/kg) blocked caffeine-induced insomnia. *: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered PFS).
[0061] FIG. 32 illustrates Entl inhibitor J4 (1.0 mg/kg) blocked caffeine-induced increase of wakefulness. *: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered PFS).
[0062] FIG. 33 illustrates Entl inhibitor J4 (6.0 mg/kg) blocked caffeine-induced increase of wakefulness. *: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered PFS).
[0063] FIG. 34 illustrates Entl inhibitor J4 (12 mg/kg) blocked caffeine-induced increase of wakefulness. *: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered PFS).
[0064] FIG. 35 illustrates the alterations of NREM sleep within ZT 1-3 after administration of
1 mg/kg (A), 6 mg/kg (B) and 12 mg/kg (C) of Entl inhibitor J4 in caffeine-induced insomnia.
Oral Entl inhibitor J4 dosages of 1 mg/kg, 6 mg/kg, and 12 mg/kg were randomly administered in the experimental protocol. The data are expressed as mean ± SEM. 1 mg/kg Entl inhibitor versus control,
< 0.05; 12 mg/kg Entl inhibitor versus control, *p < 0.05.
[0065] FIG. 36 illustrates that cage exchange cause an immediate stress response of sleep decreases and a consequent acute insomnia. *: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered PFS)
[0066] FIG. 37 illustrates the effects of cage exchange on REM sleep.
[0067] FIG. 38 illustrates the effects of cage exchange on wakefulness. *: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered PFS).
[0068] FIG. 39 illustrates the alterations of NREM sleep during (A) 1 -3 hours of light after cage-exchange, (B) 9-12 hours of light, and (C) 13-18 hours after cage-exchange. *: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered PFS).
[0069] FIG. 40 illustrates oral administration of 1.0 mg/kg Entl inhibitor J4 blocked cage exchange-induced acute insomnia (decreases of NREM sleep). *: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered PFS).
[0070] FIG. 41 illustrates oral administration of 6.0 mg/kg Entl inhibitor J4 blocked cage exchange-induced acute insomnia (decreases of NREM sleep). *: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered PFS).
[0071] FIG. 42 illustrates oral administration of 12 mg/kg Entl inhibitor J4 blocked cage exchange-induced acute insomnia (decreases of NREM sleep). *: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered PFS).
[0072] FIG. 43 illustrates the effects of 1.0 mg/kg Entl inhibitor J4 on REM sleep with cage exchange.
[0073] FIG. 44 illustrates the effects of 6.0 mg/kg Entl inhibitor J4 on REM sleep with cage exchange.
[0074] FIG. 45 illustrates the effects of 12 mg/kg Entl inhibitor J4 on REM sleep with cage exchange.
[0075] FIG. 46 illustrates the effects of 1.0 mg/kg Entl inhibitor J4 on wakefulness with cage exchange. *: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered PFS).
[0076] FIG. 47 illustrates the effects of 6.0 mg/kg Entl inhibitor J4 on wakefulness with cage exchange. *: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered PFS).
[0077] FIG. 48 illustrates the effects of 12 mg/kg Entl inhibitor J4 on wakefulness with cage exchange. *: indicates a statistically significant difference (p < 0.05) compared to the control group (orally administered PFS).
[0078] FIG. 49 illustrates the alterations in NREM sleep within ZT 1-3, ZT9-12 and ZT13-18 after administrations of 1 mg/kg (A), 6 mg/kg (B) and 12 mg/kg (C) of Entl inhibitor J4. Oral Entl inhibitor J4 doses of 1 mg/kg, 6 mg/kg and 12 mg/kg were given randomly in the experimental protocol. Data are expressed as mean ± SEM. Cage-exchange versus control, *p < 0.05; 1, 6 and 12 mg/kg Entl inhibitor J4 versus cage-exchange, #p < 0.05.
[0079] FIG. 50 illustrates the effects of vehicle and 0.5 % DMSO on NREM sleep. The icv injection time was indicated by an arrow. N.S. = no significance.
[0080] FIG. 51 illustrates the effects of vehicle and 0.5 % DMSO on REM sleep. The icv injection time was indicated by an arrow. N.S. = no significance.
[0081] FIG. 52 illustrates the effects of vehicle and 0.5 % DMSO on wakefulness. The icv injection time was indicated by an arrow. N.S. = no significance.
[0082] FIG. 53 Administration of Entl inhibitor J4 (6 mg/kg) increased NREM sleep as previous results. *: indicates a statistically significant difference (p < 0.05) compared to the control group (baseline).
[0083] FIG. 54 illustrates the effect of Entl inhibitor J4 (6 mg/kg) on REM sleep. *: indicates a statistically significant difference (p < 0.05) compared to the control group (baseline).
[0084] FIG. 55 illustrates administration of Entl inhibitor J4 (6 mg/kg) decreased wakefulness as previous results. *: indicates a statistically significant difference (p < 0.05) compared to the control group (baseline).
[0085] FIG. 56 illustrates DPCPX blocked Entl inhibitor J4-induced increases of NREM sleep. &: indicates a statistically significant difference (p < 0.05) compared to the control group (oral 1% HPβCD with icv injection DMSO).
[0086] FIG. 57 illustrates DPCPX blocked Entl inhibitor J4-induced decreases of NREM sleep. &: indicates a statistically significant difference (p < 0.05) compared to the control group (oral 1% HPβCD with icv injection DMSO).
[0087] FIG. 58 illustrates SCH58261 blocked Entl inhibitor J4-induced increases of NREM sleep. The dose of oral Entl inhibitor J4 was 6 mg/kg and HPβCD was 1%; icv injection DMSO was 5% and SCH58261 was 5 pg/lpl. Data are expressed as mean ± SEM. vehicle control versus Entl inhibitor J4 + DMSO, #p < 0.05; Entl inhibitor J4 + DMSO versus Entl inhibitor + SCH58261, &p < 0.05.
[0088] The data are expressed as mean ± SEM. Aβ + STZ versus control, *p < 0.05 and *** p < 0.001. Aβ + STZ versus Ap + STZ Entl(i), ##p < 0.01 and ###p < 0.001. N.S. = no significant differences.
DETAILED DESCRIPTION
[0089] In one embodiment, a method of treating schizophrenia is provided, including administrating to a subject a compound of formula (I), (II) or (III):
a pharmaceutically acceptable salt thereof, or a composition thereof, wherein X is halogen.
[0090] In another embodiment, the compound may be selected from N6 -[(3-halothien-2- yl)methyl] adenosine, N6 -[(4-halothien-2-yl)methyl]adenosine, and N6 -[(5-halothien-2-
yl)methyl]adenosine. Preferably, the compound is N6 -[(5-iodothien-2-yl)methyl]adenosine, N6 -[(4-iodothien-2-yl)methyl] adenosine, M’-[(3-iodothien-2-yl)methyl]adenosine, N6 -[(5- bromothien-2-yl)methyl]adenosine (also called “JMF3464” or “J4”), N6 -[(4-bromothien-2- yl)methyl]adenosine, N6- [(3 -bromo thien-2-yl)methyl]adenosine, N6 - [(5-chlorothien-2- yl)methyl]adenosine (also called “JMF3818”), N6 -[(4-chlorothien-2-yl)methyl]adenosine, N6 -
[(3-chlorothien-2-yl)methyl]adenosine, or a combination thereof.
[0091] In another embodiment, the compound may be selected from N6-[(2-halothien-3-yl)methyl] adenosine, N6 -[(4-halothien-3-yl)methyl] adenosine, and N—6 [(5-halothien-3- yl)methyl]adenosine. Preferably, the compound is N6 -[(2-iodothien-3-yl)methyl]adenosine, N6 -[(4dodothien-3-yl)methyl]adenosine, N6 -[(5-iodothien-3-yl)methyl)adenosine, N6 -[(2- bromothien-3-yl)methyl]adenosine, N6- [(4-bromothien-3 -yl)methyl] adenosine, N6 -[(5- bromothien-3 -yl)methyl] adenosine N6 -[(2-chlorothien-3-yl)methyl]adenosine, N6 -[(4- chlor othien- 3 -y l)methy 1] adenos ine. or N6 -[(5-hlorothien-3-yl)methyl]adenosine, or a combination thereof.
[0092] In one embodiment, a therapeutically effective amount of the compound is 0.5-15 mg/kg, preferably 1-12 mg/kg. In one embodiment, the compound, a pharmaceutically acceptable salt thereof, or a composition thereof is administered by an oral, nasal, intravenous, intramuscular, subcutaneous, intraperitoneal or topical route.
[0093] Embodiment 1
[0094] Materials and Methods
[0095] In the present invention, a sporadic AD (sAD) mouse is established by the unilateral intrahippocampal (ih) microinjection of Aβ aggregates (Aβ1-42) and intracerebroventricular (icv) administration of streptozotocin (STZ), a glucosamine-nitrosourea compound, and is used to assess the efficacy of a novel Entl inhibitor, J4, on the levels of nitric oxide (NO), cleaved caspase 3 and phosphorylated H2A histone family member X (y-H2AX), the activity of nuclear DNA-dependent serine/threonine protein kinase (DNA-PKcs), the alleviation of cholinergic neuronal loss in the medial septum-diagonal band of Broca, and the improvement of cognitive deficits. The effects of Entl inhibitor J4 on sAD-induced sleep disruptions were also investigated.
[0096] Animals
[0097] Ten-week-old male wild-type C57BL/6 mice (BioLASCO Taiwan Co., Ltd) weighing between 25-28 g, were used throughout the study. The mice were kept in plastic cages, and were individually housed in a standalone and soundproof chamber. Food and water were free to access. The mice were maintained in a room with constant temperature (23±1°C) with a relative humidity of 50-60% and an automatically controlled 12:12 light/ dark cycle. All procedures and animal care were performed in accordance with the Institutional Animal Care and Use Committee of National Taiwan University.
[0098] Implantation of electroencephalography (EEG)
[0099] 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 onto a stereotaxic apparatus. The scalp was incised and the head position was adjusted to keep bregma in horizontal plane. Then, the connective tissue was wiped with sterilized cotton until the bregma could be clearly seen. To record the EEG signal, the custom-made wire-wrapping-wire 30 AWG electrodes were inserted through the skull and placed above the cortex in the frontal and occipital lobes. Subsequently, the cannulae and EEG electrodes were fixed on the skull with dental cement (Tempron, GC Co., Tokyo, Japan).
[0100] EEG recording and sleep analysis
[0101] Five days after recovery from the surgery, the mice were kept in an individual chamber and connected to recording cables for 2 days for habituation before recordings. The data for baseline were obtained from the first 24 hours before drug treatment, which was used to compare with the data in the next recording on day 24th after the last drug administrations. The EEG signals were filtered and amplified to 10,000-folds by an amplifier (Coulboum Instruments, Lehigh Valley, PA, USA; model V75-01), and then manually scored each 12-s epochs from the 12-h recording using a data acquisition software ICELUS (M. R. Opp, University of Michigan). The vigilance states were defined into three states: wakefulness (low EEG amplitude and higher frequency), non-rapid eye movement sleep (NREM, large EEG amplitude with delta power greater than theta power) and rapid eye movement sleep (REM, low EEG amplitude along with an increase in theta power).
[0102] Experimental procedure
[0103] Animals were randomly divided into four groups: the sham control, vehicle control, STZ+Aβ1-42, STZ+Aβ1-42_ENTl(i). Mice in the sham group had administered 0.9% PFS into ventricle and dorsal hippocampus (dHPC; 1 pl per injection site). In the STZ+ Aβ1-42 group, mice were infused with icv injection of STZ (3 mg/kg) and ih injection ofAβ1-42 (1 pg/pl) on day 7th to 10th. The ENT1 inhibitor (6 mg/kg) or its vehicle (1% HPβCD) were administered for 18 days (between days 7th and 24th). Fourteen days after the last icv and ih injection along with oral administration of ENT 1 inhibitor, the mice were subjected to assess the spontaneous locomotor activity and anxiety level by the open field test (OFT). To evaluate the cognitive function, the Morris water maze (MWM) and novel object recognition (NOR) task were employed. EEG signals were recorded to investigate the alteration of sleep-wake activity. Right after the behavioral assay and sleep recording, the mice were sacrificed and used for the biochemical research and immunofluorescence assay (IFA).
[0104] To test the effects of Entl inhibitor J4 on physiological sleep-wake activity, the Entl inhibitor J4 at three different doses, 1, 6 and 12 mg/kg, were orally administered at either the beginning of the dark period or the beginning of the light period, and sleep-wake activities were recorded for 24 hours.
[0105] To determine the effect of Entl inhibitor J4 on caffeine-induced insomnia, caffeine was ip injected at the beginning of the light period and the Entl inhibitor J4 in three different doses (1, 6 and 12 mg/kg) were orally administered 3 hours before the light period, and the sleepwake activity was recorded for 24 hours.
[0106] To evaluate the effect of Entl inhibitor J4 on stress-induced insomnia, the cage exchange was performed at the beginning of the light period and the Entl inhibitor J4 in three different doses (1, 6 and 12 mg/kg) were orally administered 3 hours before the light period, and the sleep-wake activity was recorded for 24 hours.
[0107] Novel object recognition task (NOR task)
[0108] This behavioral task was talcing place in a white acrylic open box (40x40x45 cm) and divided into three phases: habituation, training and testing. In the initial day, the mice were allowed to freely explore the apparatus for 15 min (used as OFT) to become familiar with the
surroundings. No objects were placed in the box during the habituation phase. Twenty-four hours later, the two identical objects (Al and A2) were arranged in a diagonal position and were allowed to explore for 10 min. After the training phase, mice were placed back into the home cage, rested within the inter trial interval (90 min) and prepared for the testing phase. To assess the ability of memory in mice, one of the familiar objects was replaced by a novel object (B) and allowed to explore for 5 min and acquired the short-term memory (STM). Long-term memory test was conducted 24 h after training, and the 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. Exploration behavior was defined as sniffing or touching the objects with the nose or forepaws < 2 cm away from it. The discrimination index (DI) was calculated for each mouse and was presented as the ratio of subtracting the time spent in novel one (B or C) from familiar one (A) and divided the total exploration time in both objects (DI=([B or C]-A)/ ([B or C] +A)).
[0109] Morris water maze (MWM)
[0110] The task was conducted in a circular pool (153 cm in diameter) filled with water (25±1°C), which mixed with non-toxic white paint and turned transparent water to opaque, and the pool was divided into four quadrants. An escape platform (10 cm in diameter) was placed into quadrant 4 (target quadrant) and submerged 2 cm below the surface of water. The visual cues were pasted on the edge of the pool to provide the orientation for mice. In the training phase, all the mice were given three trials per session for five consecutive days (days 19, 20, 21, 22, 23), and allowed them to search the hidden platform for 120 s in each trial. After they climbed on the platform, the mice had left on it for 30 s before returning to the home cage. Once the mice failed to find the platform, they would be gently guided and allowed to stay on platform for 30 s. Twenty-four hours after the last training trial, the probe test (on day 24) was given by removing the escape platform from the target quadrant, and the mice were allowed to freely swim for 120 s. The escape latency, meaning the time taken to reach the platform in the training phase was evaluated. Time spent in the target quadrant and the number of times passed through the original position of the platform in the probe test were measured by video tracking
software (EthoVision XT version 14.0.1322, Noldus Information Technology by the Netherlands).
[0111] Results
[0112] The hallmarks of sAD established by streptozotocin (STZ) and Aβ
[0113] The hallmarks of sAD were observed in the 14th day after 4 times of administering N6 [3 aggregates by the unilateral ih microinjection and STZ by the icv administration in 4 consecutive days. The accumulation of N6 [3 plaques in mice was measured by the optical density after receiving icv-STZ and ih- N6[3, indicating that Aβ plaques widely distributed in the CAI, CA3 and hilus of the hippocampus as shown in FIG. IB when compared with the PFS- treated control mice (FIG. 1A). Unpaired student t-test comparisons showed the statistically significant difference in amyloid-beta deposition (the ratio of Ap positive area/tissue area) between the control group (~ 0 pixel) and the group received icv-STZ with ih-Aβ (0.045 ± 0.011 pixels; p < 0.001 vs. control) as shown in FIG. 1C.
[0114] The levels of phosphorylated tau protein at the residue Ser404 in the 14th day after receiving four-time administrations of icv-STZ and ih-Aβ were measured, and the result indicated that the ratio of phospho-tau/total tau in the hippocampus elicited significant increase from the control of 0.81 ± 0.08 to 2.27± 0.31 (p < 0.001, FIGs. ID and IE). However, the protein level of total tau in the hippocampus was not altered after treatment with icv-STZ and ih-Aβ (FIG. IF). These results demonstrated that four-time administrations of icv-STZ and ih-Aβ could establish accumulations of Ap and phosphorylated tau protein in brains to simulate the sAD in mice.
[0115] Effects of Entl inhibitor J4 on cholinergic neuronal loss in the medial septumdiagonal band of Broca (MSDB) after icv-STZ and ih-Aβ
[0116] The mice treated with icv-STZ and ih-Aβ exhibited 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) when compared with the neuron numbers of 60.11 ± 2.98 obtained from the control group (FIGs. 2A, 2B, 2E). The sAD mice treated with Entl inhibitor J4 elucidated that the cholinergic neuronal loss could be prevented, and the number of cholinergic neurons in MSDB maintained as 51.11 ± 1.15 (vs.
Ap + STZ, p < 0.001; FIGs. 2D and 2E). Since J4 was dissolved in the vehicle of 1% 2- Hydroxypropyl-beta-cyclodextin (HPβCD), it was also to be determined whether 1% HPβCD affects cholinergic neuronal loss, and the result indicated that HPβCD exhibited no effect as shown in FIGs. 2C and 2E. This result elicited that elevation of extracellular adenosine concentrations by ENT inhibitor is beneficial to the basal forebrain cholinergic system.
[0117] Effects of Entl inhibitor J4 on the impairments of recognition memory in icv- STZ and ih-Aβ-induced sAD mice
[0118] Based on the numerous abnormalities occurring in the hippocampus and the cholinergic neuronal loss in the MSDB, the behavioral assays of novel object recognition (NOR) test and Morris water maze (MWM) test were employed to determine whether the cognitive functions were impaired.
[0119] As the sAD was established by icv-STZ and ih-Aβ, the short term and long term memory were assessed in the first and second testing phases of the NOR test, which was described in the following method section.
[0120] Please refer to FIG. 3. The result demonstrated that icv-STZ and ih-Aβ-induced sAD mice were unable to discriminate between the familiar and novel objects, and spent more time exploring the familiar object when compared with its littermate in the control group. The discrimination indexes obtained from the sAD mice were -0.27 ± 0.12 (p < 0.01, vs. control, one-way ANOVA with post hoc comparison) for the short-term memory (STM) and -0.2 ± 0.06 (p < 0.05, vs. control, one-way ANOVA with post hoc comparison) for the long-term memory (LTM), which were significantly declined when compared to those obtained from the control in the STM (0.27 ± 0.08) and in the LTM (0.21 ± 0.15). Whereas oral-administration of the Entl inhibitor J4 produced a significant improvement in discriminating ability between the familiar and novel objects in these sAD mice. The discrimination indexes obtained from the sAD mice received Entl inhibitor J4 were 0.49 ± 0.1 for the STM (p < 0.001 vs. Ap + STZ, one-way ANOVA with post hoc comparison) and 0.62 ± 0.11 for the LTM (p < 0.001 vs. Ap + STZ, one-way ANOVA with post hoc comparison). Oral administration of vehicle HPβCD in control mice did not alter the discrimination indexes no matter in the STM and LTM. HPβCD vehicle also exhibit no significant improvement in the decline of discrimination index
obtained from the sAD mice. This result elucidated J4 prevented the cognitive decline in the icv-STZ and ih-Aβ-induced sAD mice, suggesting that Entl inhibitor J4 improved the recognition memory in the sAD mice.
[0121] Effects of Entl inhibitor J4 on the impairments of spatial memory in icv-STZ and ih-Aβ-induced sAD mice
[0122] The learning of spatial memory was also determined by the heat map of swimming traces and the time staying in quadrant II with or without a hidden platform in the Morris water maze (MWM).
[0123] Please refer to FIG. 4A first. The escape latency, meaning the time taken to reach the platform in the training phase, was evaluated. The result demonstrated that mice treated with ih-Aβ and icv-STZ took significantly longer time to reach the hidden platform than its littermate in the control group on the 2nd, 4th and 5th days, demonstrating the learning impairment. Interestingly, treatment of Entl inhibitor J4 enabled sAD mice to significantly improve their ability to find the hidden platform during the 2nd to 5th days. Oral administration with the HPβCD did not exhibit any effect on the escape latency in the sAD mice.
[0124] Please refer to FIG. 4B. The control mice exhibited the preference to stay in the target quadrant (quadrant II), where the hidden platform existed during the training sessions, in the probe test when the hidden platform was removed. Mice treated with HPβCD did not change the preference for mice to stay in quadrant II in the probe test. Mice treated with ih-Aβ and icv-STZ lost the preference to stay in quadrant II, indicating the impairment of spatial memory'; while J4 could reverse the memory impairment in the sAD mice.
[0125] Please further refer to FIG. 4C. The time for sAD mice to linger in the target quadrant was significantly decreased to 14.04 ± 2.35 sec when compared to 31.52 ± 3.08 sec obtained from the control mice (sAD vs. control, p < 0.01). Administration of the vehicle HPβCD did not exhibit any improvement on the spatial memory in sAD mice. The Entl inhibitor J4 significantly improved the time spent in the target quadrant to 37.52 ± 5.75 sec (p < 0.001 vs. sAD mice) when compared to that obtained from sAD mice without any treatment. However, there were no significant changes among the control mice, sAD mice and sAD mice treated
with either vehicle or J4, suggesting that Entl inhibitor J4 improved the spatial memory in the sAD mice.
[0126] Effects of the Entl inhibitor J4 on the expression of NO in the sAD mice
[0127] Mice treated with icv-STZ and ih-Aβ would induce oxidative stress and subsequently cause DNA damage and neuronal apoptosis as the cholinergic neuronal loss in the MSDB was observed. Please further refer to FIG. 5. The NO levels were evaluated by the amounts of the nitrite. The concentrations of nitrite were significantly increased from 19.56 ± 1.72 pM and 18.04 ± 1.62 μM, respectively, obtained from the control and vehicle HPβCD to 27.82 ± 2.23 pM (p < 0.05 vs. control or vehicle HPβCD) in the hippocampus after treated with icv-STZ and ih-Aβ. The sAD mice treated with J4 exhibited an attenuation of nitrite levels to 18.33 ± 1.45 pM when compared with that of sAD mice per se (p < 0.01), indicating Entl inhibitor J4 blocked the oxidative stress in sAD.
[0128] Effects of the Entl inhibitor J4 on DNA damage and apoptosis in the sAD mice
[0129] The abnormal increase of NO in the hippocampus may further cause DNA damage and cell apoptosis. Please refer to FIG. 6A. The results indicated that the ratio of y-H2AX/a- tubulin, the indicator of DNA double strand breaking (DSB) marker, was statistically significantly increased in the hippocampus when the sAD was established after treated with icv-STZ and ih-Aβ. The ratio of y-H2 AX/a-tubulin was increased from 0.14 ± 0.03 and 0.2 ± 0.03, respectively, obtained from control and vehicle HPβCD to 0.58 ± 0.06 (p < 0.001). Oral gavage of the Entl inhibitor J4 significantly attenuated the ratio of y-H2 AX/a-tubulin in the hippocampus to 0.37 ± 0.06 (p < 0.05 vs. sAD mice).
[0130] Please further refer to FIG. 6B. A similar observation was also found in the expression of cleaved caspase 3, the indicator of cell apoptosis. The ratio of cleaved caspase 3/a-tubulin was increased from 0.3 ± 0.06 and 0.25 ± 0.04, respectively, obtained from control and vehicle HPβCD to 0.69 ± 0.03 (p < 0.001). While oral gavage of the Entl inhibitor J4 significantly reduced the ratio of cleaved caspase 3/ a-tubulin to 0.69 ± 0.03 (p < 0.001 vs. sAD mice) in the hippocampus. Taken together, augmentation of the extracellular adenosine by Entl inhibitor J4 can relieve the DNA damage and apoptosis in sAD mice.
[0131] Effects of the Entl inhibitor J4 on the activity of DNA-PKcs in the sAD mice
[0132] According to elevation of DNA DSB marker and apoptosis in the sAD mice, the activity of major enzyme DNA-PKcs that mediates the NHEJ pathway to repair double strand breaks in DNA is next evaluated. Please further refer to FIG. 6C. The active form of DNA-PKcs is determined by the phosphorylation at Thr2609. The results from Western blot analysis revealed that the ratio of phosphorylated-DNA PKcs/DNA PKcs was decreased from 1.05 ± 0.13 and 1.02 ± 0.05, respectively, obtained from the control and vehicle HPβCD to 0.72 ± 0.06 (p < 0.05) after treated with icv-STZ and ih-Aβ. Administration of Entl inhibitor J4 significantly increased the ratio of phosphorylated-DNA PKcs/DNA PKcs back to 1.02 ± 0.05 (p < 0.05 vs. sAD mice). However, the levels of total DNA PKcs were not altered among these groups.
[0133] Effects of the Entl inhibitor J4 on sAD-induced sleep disruptions
[0134] Since sleep disruptions are commonly found in AD patients, the alterations of sleep- wake activity and sleep architectures after administrations of icv-STZ and ih-Aβ and the effects of J4 were determined. The results revealed that NREM sleep during the first three hours (zeitgeber time [ZT] 13- 15) of the dark period was significantly increased from 11.04 ± 3.92 % obtained from the control to 38.83 ± 2.54 % (p < 0.001, one-way ANOVA with post hoc comparison, FIGs. 7A and 8A) in the sAD mice induced by icv-STZ and ih-Aβ, while the amount of NREM sleep during ZT’3-5 of the light period in the following lightdark cycle was suppressed from 57.69 ± 3.56 % obtained from the control to 35.16 ± 3.52 % (p < 0.05, one- way ANOVA with post hoc comparison, FIGs. 7A and 8B). In addition, REM sleep during ZT’ 1-11 was significantly decreased from 10.7 ± 0.69 % obtained from the control to 3.04 ± 0.49 % (p < 0.001, one-way ANOVA with post hoc comparison, FIGs. 7B and 8C). Wakefulness exhibited a mirror effect in response to the sleep alteration. Wakefulness was markedly decreased from 88.31 ± 4.24 % to 59.02 ± 2.9 % (p < 0.05, one-way ANOVA with post hoc comparison) during ZT13-15 (FIG. 8D) and significantly increased from 23.81 ± 3.11 % to 46.68 ± 2.81 % (p < 0.05, one-way ANOVA with post hoc comparison) during ZT’3-9 in the sAD mice (FIGs. 7C and 8E).
[0135] Oral administration of J4 reversed the enhancement of NREM sleep during ZT13-15 the back to 17.87 ± 3.96 % (p < 0.05 vs. Ap + STZ, one-way ANOVA with post hoc
comparison, FIGs. 7D and 8 A) and normalized the suppression of NREM sleep during ZT’3- 5 to 63.75 ± 4.11 % (p < 0.05 vs. Aβ + STZ, one-way ANOVA with post hoc comparison, FIGs. 7D and 8B) in the sAD mice. The sAD-induced suppression of REM sleep during ZT’ 1- 11 was blocked by J4, and the amount of REM sleep after administering J4 was 8.91 ± 0.56 % (p < 0.05 vs. Aβ ± STZ, one-way ANOVA with post hoc comparison, FIGs. 7E and 8C). J4 also respectively reversed the decreased wakefulness during ZT13-15 and enhancement of wakefulness during ZT’3-9 back to 82.12 ± 4. 16 % (p < 0.05 vs. Aβ ± STZ, one-way ANOVA with post hoc comparison, FIGs. 7F and 8D) and 28.38 ± 3.0 % (p < 0.05 vs. Aβ + STZ, one- way ANOVA with post hoc comparison, FIGs. 7F and 8E).
[0136] Analyzing the alterations of sleep architecture revealed that the reduction of NREM sleep during the light period after treating with icv-STZ and ih-Aβ was primarily due to the decrease of NREM bout duration, although the bout number of NREM sleep was increased (FIGs. 8F and 8G). Application of Entl inhibitor J4 could successfully block the reduction of NREM bout duration and reverse the enhancement of NREM bout number in the sAD mice (FIGs. 8F and 8G). The reduction of REM sleep during the light period was due to the decreases in both the bout duration and the bout number of REM sleep in the sAD mice, and J4 blocked these suppressions (FIGs. 8H and 81). Furthermore, the transition between vigilance states was increased in the sAD mice, suggesting sleep fragmentation, and J4 corrected it (FIG. 8J).
[0137] Discussion
[0138] Sleep problems gradually became an issue in AD. sAD is the most common neurodegenerative disorder with neuronal apoptosis, declined cognitive functions and sleep disruptions. The results according to the embodiments of the present invention revealed that the sleep architectures were significantly altered in the Ap + STZ-induced sAD mice. The affected sleep pattern exhibited that NREM sleep, REM sleep and wakefulness were not in line with the normal sleep homeostasis and accompanied with the short sleep duration. However, there were no significant differences in transitions during the light period. Although the transitions were not conclusive, a tendency of sleep fragmentation in the Ap + STZ-induced sAD mice could be observed. It can be concluded that the alterations of sleep-wake activity in
the icv-STZ and ih- Aβ-induced sAD mice are approximately similar to those sleep disruptions in AD patients.
[0139] Sleep disorder and AD exhibit even a bidirectional relationship. Decreasing the sleep time can increase the production of pathological hallmarks in AD; and abnormal aggregation of the neurotoxic proteins may result in the neuronal damage and alterations of the sleep pattern. In addition to the influence on the progression of AD. several studies have examined the relationship between sleep disturbance and memory decline. Disrupting the sharp-wave ripples and theta rhythms in the hippocampus respectively during NREM and REM sleep are correlated to the impairment of memory consolidation and storage.
[0140] The results of the present invention indicated that treatment with the Entl inhibitor J4 normalized the levels of nitric oxide, cleaved-caspase 3 and phosphorylated H2A histone family member X (y-H2AX), and increased activities of nuclear DNA-dependent serine/threonine protein kinase (DNA-PKcs) through the non-homologous end joining (NHEJ) pathway to repair double-strand breaks in DNA. J4 also alleviated the loss of cholinergic neurons in the medial septum-diagonal band of Broca, and further improved cognitive deficits. [0141] Further, the sAD mice increased NREM sleep during dark period and decreased NREM and REM sleep during light period, while the elevation of extracellular adenosine exhibited beneficial for the homeostatic sleep. In other words, treatment with the Entl inhibitor in the present invention normalized the disrupted sleep pattern in the sAD mice, suggesting adenosine augmentation evoked by the Entl inhibitor has positive effects on regulating homeostatic sleep. Adenosine is a sleep promoting substance, and blocking the Entl results in the increased extracellular adenosine. Therefore, it can be assumed that it would enhance the binding between the adenosine receptor and adenosine, which further regulates sleep in the CNS. In conclusion, the Entl inhibitor J4 may be potential for sAD treatment.
[0142] Embodiment 2
[0143] Materials and Methods
[0144] Animals and Implantation of EEG
[0145] Male C57BL/6 mice (6-8 weeks old; BioLASCO Taiwan Co., Ltd) and GAD67-GFP mice were used in this research. The original strain of GAD67-GFP mice was B6 (provide by
Dr. M.Y. Ming, National Taiwan university). Mice were anesthetized with Zoletil (10 mg/kg, Carros, France) and xylazine (12 mg/kg, Sigma- Aldrich, USA). Two EEG electrodes have been implanted in the frontal and parietal lobes of the brains and an additional intracerebroventricular (icv) guide cannula was implanted into the ventricle in male C57BL/6 mice. The icv guide cannula was implanted with coordinates: AP, -0.2 mm from bregma; ML, -1 mm; DV, -2.1 mm. After the implantation, they are fixed on the skull using dental acrylic (Tempron, GC Co., Tokyo, Japan). Then analgesic ibuprofen (0.4 g/250 ml, Yung Shin Pharm. Ind. Co., Ltd) was added into the drinking water after surgery for seven days. Mice were recovered for seven to ten days and mice were habituated by daily handling. The administrations of pyrogen-free saline (PFS) by icv and oral gavage were timed to coincide with scheduled experimental administrations to reduce the extra stress effect before the experiment. Mice were hosted in the individual cage with the constant temperature at 23-24°C in the 12:12h cycle laboratory animal room. All food and water were provided ad libitum. All these procedures were approved by the National Taiwan University Institutional Animal Care and Use Committee (IACUC).
[0146] EEG recording and sleep analysis
[0147] The EEG signals were amplified by an amplifier (Coulbourn Instruments, Lehigh Valley, PA, USA; model V75-01). Analog bandpass filtering between 0.1 and 40 Hz was applied to the EEG, and a gain of 10,000 was added. These filtered EEG signals were fed into a 128 Hz sample rate analog-to-digital converter (NI PCI-6033E, National Instrument, Austin, TX, USA). The digital EEG signals were preserved as binary files for use in further sleep analysis. The sleep-wake activity were recorded and manually analyzed with a 12-s epoch by a custom software ICELUS (M. R. Opp, University of Michigan) written in Lab View for Windows (National Instruments). The vigilances of wakefulness, NREM sleep, and REM sleep were categorized according to the following criteria in rodents. During wakefulness, low amplitude and high-frequency EEG waves were presented. A synchronized large-EEG amplitude dominating with 0.5-4.0 Hz delta waves were shown during NREM sleep, and a dominant 6.0-9.0 Hz theta waves were displayed in REM sleep. Moreover, the sleep
architectures, including bout numbers, bout duration (min) and stages transitions, were also assessed.
[0148] Substances
[0149] Three different concentrations of Entl inhibitor J4 (1.0, 6.0, 12.0 mg/kg) were dissolved in 1% 2-Hydroxypropyl-beta-cyclodextin (HPβCD) solution for oral gavage to investigate one-time effect on physiological sleep. A low dose of caffeine (10 mg/kg, Sigma- Aldrich, USA) was used to induce insomnia. Both the AiR antagonist, 8-cyclopentyl-l,3- dipropylxanthine (DPCPX; Sigma-Aldrich, USA) and the A2AR antagonist, 5-amino-7-([3- phenylethyl)-2-(8-furyl) pyrazolo[4,3-e]- 1 ,2,4-triazolo[ 1 ,5-c] pyrimidine (SCH58261 ; Tocris), were dissolved in 0.5 % DMSO in doses of 5 pg/ 1 pl for the icv administration. PFS and 0.5 % DMSO were used as vehicle control.
[0150] Experimental procedure for testing drug concentration gradients
[0151] After recovery on postoperative days 7-10, the EEG recording electrodes were implanted into the skull of the experimental mice, and the mice were given 0.5 ml of PFS by oral gavage 30 minutes before the switch of light-to-dark period. Mice were adapted to the experimental operation for ten consecutive days. After the oral administration of PFS on the postoperative days 17-20, the control group was started with a 24-hour recording, and different concentrations of Entl inhibitor J4 (1.0, 6.0, 12.0 mg/kg) were followed and randomly administered orally, and the same 24-hour recording was done for the control group (the interval between doses is 2 days). (FIG. 9)
[0152] Acute insomnia model
[0153] In these experiments, we used two different ways to create insomnia in mice:
[0154] Example 1: Stress-induced insomnia model
[0155] The surgical and acclimatization protocols were identical to the sections for testing drug concentration gradients. Next, 24 hours were recorded after oral administration of PFS 3 hours before the light period on postoperative days 17-20. In the control group, acute insomnia was induced by cage change during the dark/light transition and the sleep variation were recorded. Subsequently, different concentrations of J4 ( 1.0, 6.0, 12.0 mg/kg) were given orally at random
after 48 hours (each dose interval was 2 days), and finally, the sleep changes were analyzed and compared sequentially (FIG. 10).
[0156] Example 2: Caffeine-induced insomnia model
[0157] The surgical and habilitation regimens were the same as described above. One of the differences from the above experiments is that on postoperative days 17-20, the baseline group will perform a 24-hour recording after oral administration of PFS 3 hours before the light period; the control group will perform an intraperitoneal injection of caffeine 30 minutes before the light period after oral administration of PFS to simulate the insomnia response induced by receiving caffeine. A 24-hour recording was started in the control group, and 48 hours later, different concentrations of Entl inhibitor J4 (1.0, 6.0, 12 mg/kg) were randomly added orally, and the same 24-hour recording was performed in the control group. (Each dose interval was 2 days.) (FIG. 11)
[0158] Immunofluorescence assay
[0159] The rodents utilized in the experiment were kept in the same setting as the previous experiments. To help the subjects adjust to the study procedure, PFS was given orally for seven days. On the eighth day, the administration group received an oral dose of 6 mg/kg Entl inhibitor J4 or PFS (control group). At the fifth hour following oral delivery, the experimental mice’s brains were perfused with 4% paraformaldehyde and removed. 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 the fifth hour. After the mice showed no signs of pain, the thorax was cut and open. In order to begin PFS perfusion, the perfusion needle (also known as a butterfly needle) was placed into the left ventricle of the experimental mice. The experimental mouse's whole blood was then substituted with PFS (depending on whether the liver was bloodless), and thereafter 4% paraformaldehyde (P6148, Sigma- Aldrich, USA) was added. The entire brain was swiftly removed and impregnated with 4% PFA at room temperature for 6 hours after the 4 % PFA had replenished throughout the entire body of the mouse (there was no longer any torso pulsation from myofibrillar degeneration). The entire brain was instantly removed and incubated for 6 hours at room temperature with 4% PFA. The whole brain was then replaced with a 20 % glycerol (Sigma- Aldrich, USA) in 0.1 % PB
solution at 4 °C for 24-48 hours to avoid physical damage during the freezing of the sections. Moreover, the brain was embedded with Optimal Cutting Temperature (O.C.T.) compound (Sakura Finetek USA, Inc.) and stored at -80°C for the frozen section. After the antifreeze treatment, brain tissue was sectioned using the method of dry ice section (The machine provided by Dr. Yen, C. D., Department of Life Science, National Taiwan University), and brain tissue was sectioned from 0.10 mm posterior to 0.14 mm anterior to the bregma (ventrolateral preoptic nucleus in adult mice). Next, the sections were rinsed three times with PBS for 5 minutes each and then incubated with PBS containing 0.3 % Triton X-100 (PBST) and shaken for 15 minutes at room temperature. To reduce nonspecific binding, brain sections were blocked using 3 % normal goat serum (NGS; Jackson Immunoresearch) dissolved in PBS. At the end of this phase, slices were stained with the following primary antibody: anti-fos (1.T000; abl90289; abeam) and incubate for 16 hours at 4°C. At the end of the incubation, slices were rinsed 3 times with PBS for 5 minutes each and then incubated for 1 hour with the corresponding secondary antibody: goat anti-rabbit IgG H&L Alexa Fluor ®594 (1 :500; ab150080; abeam). Finally, the slices were rinsed three times with PBS for 5 minutes each.
[0160] At the end of the above steps, in order to more easily visualize the cells, the nuclei were stained with DAPI (D5242, Sigma-Aldrich) in PBS solution at a concentration of 100 ng/mL for 10 minutes, and the slices were rinsed six times with PBS for 10 minutes each. After airdrying to remove excess water, each brain slice was covered with a 0.17 mm thick coverslip (AP-0810401, Sigma- Aldrich) and few drops of fluoromountTM aqueous mounting media (F4680, Sigma-Aldrich). The Olympus 1X83 inverted fluorescence microscope was used to capture the fluorescent images (provided by Dr. Pei-Hsueh Tsai, Department of Veterinary Medicine, National Taiwan University).
[0161] Statistical analysis
[0162] The mean and standard error of the mean (SEM) were used to represent all of the data, and SPSS was used to analyze the results. The independent sample /-test and one-way ANOVA were used to calculate the difference between the groups. Based on the following criterion, the statistical significance was determined. *: P<0.05 (significant).
[0163] Results
[0164] Effects of oral Entl inhibitor J4 on physiological sleep in experimental mice during the light period
[0001] The oral administration of three different concentration gradients of Entl inhibitor J4 (1.0, 6.0, 12.0 mg/kg) were compared to the control group (1% HPβCD) within 24 hours of administration. The results showed no significant change in NREM sleep for 12 hours (ZT1- 12, light period) after administering the three different concentrations of J4 30 minutes before the light period (FIGs. 12-14). In contrast, there was a significant increase in NREM sleep (FIG.15) and a decrease in the proportion of wakefulness between 13 and 18 hours (ZT13-18) after oral gavage of three different concentrations of J4 (FIGs. 16-18). In addition, there was no significant change in REM-sleep after administration of the three different concentrations ofJ4 (FIGs. 19-21).
[0165] Effects of oral Entl inhibitor J4 on physiological sleep in experimental mice during the dark period
[0166] As described above, the effects of Entl inhibitor J4 on sleep were tested using the same method. Oral gavage of three different concentration gradients of J4 (1.0, 6.0, 12.0 mg/kg) versus control (1% HPβCD) during the first 30 minutes of the dark period and sleep-wake activity was recorded for 24 hours. The results showed a trend of increase NREM sleep during 1-3 hours (ZT13-15) after administration of three different concentrations of J4, but no significant difference was observed (FIG. 22). In contrast, three different doses (1.0, 6.0, 12.0 mg/kg) of J4 significantly increased NREM sleep and decreased during 4-6 hours (ZT16-18) (FIG. 22). In addition, there was no significant change in REM sleep during ZT13-18 after administration of three different concentrations of J4 (FIGs. 23-25).
[0167] Entl inhibitor J4 blocked caffeine-induced insomnia
[0168] The invention first aimed to determine whether the Entl inhibitor exhibits blockade effect on caffeine-induced insomnia after knowing the onset time of Entl inhibitor. First, IP injection of caffeine during the dark-light period produced caffeine-induced insomnia effects. Referring to the results shown in FIGs. 26-28, there was a statistically significant decrease during ZT1-2 after oral administration of caffeine when compared to those after administering 1% HPβCD and PFS. Next, the changes in the amount of NREM sleep over 24 hours in
caffeine-induced insomnia with different concentrations of Entl inhibitor J4 were further analyzed. Two doses of J4 (1 mg/kg and 12 mg/kg) significantly blocked caffeine-induced NREM sleep decrease (FIGs. 29-34) and wakefulness increase during ZT1-2 (Parts A and C of FIG. 35). The administration of 6 mg/kg J4 only showed a tendency to block caffeine-incused increase of NREM sleep and decrease of wakefulness, but there was no significant difference (Part B of FIG. 35). (Veh= vehicle)
[0169] Entl inhibitor J4 blocked stress-induced insomnia
[0170] In addition to caffeine-induced insomnia, cage-exchange was used to induce two stages of sleep effects: initial stress response and acute insomnia. First, in the group with oral PFS and the group with oral PFS and cage change at the transition between the dark and light periods, there was a statistically significant decrease of initial stress response with a NREM sleep decrease within 1-4 hours after cage change (ZT1-4), no significant difference during hours 5-9 (ZT5-9), and the acute insomnia of NREM decrease during ZT10-18. Acute insomnia symptoms lasted for 9 hours after cage change (FIGs. 36-39). Based on the results, a comparison was made between the oral administration of PFS and the oral administration of three different concentrations of J4 (1.0, 6.0 and 12.0 mg/kg) with a cage exchange. It was found that Entl inhibitor J4 blocked the acute insomnia induced by the cage exchange (FIGs. 40-49). In addition, there was no significant difference in REM sleep.
[0171] The effect of Entl inhibitor J4 on sleep was mediated by adenosine receptors
[0172] The previous results have demonstrated that oral administration of Entl inhibitor significantly increased NREM sleep and improved acute insomnia. In addition, oral administration of Entl inhibitor was also demonstrated to improve insomnia caused by caffeine. Caffeine is a non-selective antagonist of adenosine receptors for type Ai and AZA. Therefore, this experiment further demonstrated whether oral gavage of Entl inhibitor J4 has achieved sleep promotion by activating adenosine type Ai or A 2A receptors.
[0173] AiR antagonist DPCPX blocked effect of Entl inhibitor J4
[0174] To exclude the effect of icv injection, the following four treatments were compared. These four groups are as follows. Baseline group: no treatment and drug administration after habituate; Vehicle (1 % HPβCD)+DMSO group: oral administration of 1 % HPβCD and icv
injection DMSO three and a half hours after drug administration (ZT15); Entl inhibitor J4+DMSO group: oral administration of J4 and three and a half hours after dosing (ZT15) icv injection DMSO, and Entl inhibitor J4+DPCPX group: oral administration of J4 and three and a half hours after dosing (ZT15) icv-injection DPCPX (adenosine Ai receptor antagonist). The results revealed no significant changes in NREM sleep in the baseline and Vehicle+DMSO groups during the 24 hours of recording (FIGs. 50-52). J4 also increased NREM sleep and decreased wakefulness, but not REM sleep, in these group as previous results (FIGs. 53-55). Application of AiR antagonist DPCPX significantly blocked Entl inhibitor induced increases of NREM sleep and the decreases of wakefulness (FIGs. 56 and 57).
[0175] A2AR antagonist SCH58261bIocked effect of Entl inhibitor J4
[0176] Further to above, the effect of SCH58261 (adenosine 2A receptor antagonist) on Entl inhibitor J4 was performed. J4 was administered orally with icv injection SCH58261 three hours after administration (ZT15). The results showed that application of AZAR antagonist SCH58261 also significantly blocked Entl inhibitor induced increases of NREM sleep and the decreases of wakefulness, but not REM sleep. However, the decrease was not as great as in the icv injection DPCPX group, and there was still no significant difference in REM-Sleep (FIG. 58).
[0177] Discussion
[0178] The Entl inhibitor J4 efficacy in sleep
[0179] The study was conducted by oral administration of substances and undisturbed experimental mice. Entl inhibitor J4 was administered in three concentration gradients (1.0, 6.0, 12 mg/kg) 30 minutes before the mice were about to enter the active or resting phase. In the dark phase, there was a significant increase in NREM sleep for 4-6 hours after three doses of Entl inhibitor J4, while in the light phase there was a significant increase in NREM sleep for 13-18 hours after given. Taken together, J4 not only caused drowsiness in the dark phase (active phase), but also increased and prolonged NREM sleep in the light phase (sleep phase) after oral administration.
[0180] Entl inhibitor J4 improved insomnia
[0181] In this study, two models of insomnia were used: acute insomnia due to stress and insomnia due to caffeine intake. The two models were chosen because insomnia is known for many reasons, such as stress, chronic pain, lifestyle, overexcitement, or drugs such as caffeine. As a non-selective adenosine antagonist, caffeine is present in our daily life. Most people consume caffeine as a refreshing agent, but inappropriate caffeine intake can produce different degrees of sleep disturbance. In many sleep-related studies, caffeine has been used to indicate transient insomnia. In this study, administration of the Entl inhibitor J4 could reduce and block the pharmacological properties produced by caffeine. The results showed that J4 could reduce the pharmacological properties of caffeine.
[0182] Potential for Entl inhibitor J4 as hypnotic drug
[0183] Although the absorption efficiency between human and mice are different, if the above results are applied, Entl inhibitor J4 can be taken 3 hours before bedtime to help stabilize sleep during the night. Currently, there are two main types of sleeping drugs available on the market, namely benzodiazepines (BZD) and non-benzodiazepines (non-BZD). The major adverse side effects include addiction and withdrawal symptoms, which cause anxiolytic effects, muscle relaxation, and memory impairment in addition to sleep-inducing effects. More often, it may cause dependency/ abuse related problems, and muscle relaxation and memory impairment, and may be strongly associated with falls in the elderly. The effect of J4 on sleep alone is probably better than that of BZD drugs, and is similar to that of Dual Orexin Receptor Antagonists (DORAs), a new type of sleeping drug that has just hit the market, both combining with two G protein couple receptors (orexin receptor- 1 and -2). In addition to DORAs, the typical aminobutyric acid receptor agonist sedative hypnosis treatment for insomnia may offer an extra and alternative pharmaceutical strategy by targeting the adenosine receptor system. J4, on the one hand, this new compound, can affect the AiR, which is distributed throughout the brain, indirectly regulating the performance of the waking neuron, and furthermore, it can also affect the A2AR, the GABAergic neuron responsible for directly increasing NREM sleep. Thus, J4 may be potential as hypnotic drug.
[0184] The many features and advantages of the present disclosure are apparent from the detailed specification, and thus, it is 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, since numerous modifications and variations will readily occur to those skilled in the art, it is not desired to limit the present disclosure to the exact construction and operation illustrated and described, and accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the present disclosure.
[0185] Moreover, those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be used as a basis for designing other structures, methods, and systems for carrying out the several purposes of the present disclosure. Accordingly, the claims are not to be considered as limited by the foregoing description.
Claims
1. A method of treating a sleep disruption in a subject in need thereof, comprising administering to the subject a compound of formula (I), (II) or (III):
a pharmaceutically acceptable salt thereof, or a composition thereof, wherein X is halogen.
2. The method of claim 1, wherein the compound is selected from the group consisting of N6 - [(3-halothien-2-yl)methyl]adenosine, N6 -[(4-halothien-2-yl)methyl]adenosine, and N6 - [(5-halothien-2-yl)methyl]adenosine.
3. The method of claim 2, wherein the compound is selected from the group consisting of N6 - [(5-iodothien-2-yl)methyl]adenosine, N6 -[(4-iodothien-2-yl)methyl]adenosine, N6 -[(3- iodothien-2-yl)methyl]adenosine, N6 -[(5-bromothien-2-yl)methyl]adenosine, N6 -[(4- bromothien-2-yl)methyl]adenosine, N6 -[(3-bromothien-2-yl)methyl]adenosine, N6 -[(5- chlorothien-2-yl)methyl]adenosine, N6 -[(4-chlorothien-2-yl)methyl]adenosine, and N6- [(3-chlorothien-2-yl)methyl]adenosine.
4. The method of claim 1 , wherein the compound is selected from the group consisting of N6 - [(2-halothien-3-yl)methyl]adenosine, N6 -[(4-halothien-3-yl)methyl]adenosine, and N6- [(5-halothien-3 -yl)methyl] adenosine.
5. The method of claim 4, wherein the compound is selected from the group consisting of N6 -
[(2-iodothien-3-yl)methyl]adenosine, N6 -[(4-iodothien-3-yl)methyl]adenosine, N6 -[(5- iodothien-3-yl)methyl] adenosine, N6 - [(2-bromothien-3 -yl)methy 1] adenosine, N6-[(4- bromothien-3-yl)methyl]adenosine, N N6 -6[(5-bromothien-3-yl)methyl]adenosine N6 -[(2- chlorothien-3-yl)methyl]adenosine, N6 - [(4-chlorothien-3-yl)methyl] adenosine, and N6- [(5-chlorothien-3-yl)methyl]adenosine.
6. The method of claim 1, wherein a therapeutically effective amount of the compound is 0.5-15 mg/kg.
7. The method of claim 1 , wherein the compound, a pharmaceutically acceptable salt thereof, or a composition thereof is administered by an oral, nasal, 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. The method of claim 1, wherein the subject has insomnia or Alzheimer’s disease (AD).
10. The method of claim 9, wherein the insomnia is stress-induced insomnia, caffeine-induced insomnia or a combination thereof.
11. A composition for use in a method of treating a sleep disruption in a subject in need thereof, comprising administering to the subject the composition comprising a compound of formula (I), (II) or (III):
12. The composition for use of claim 11, wherein the compound is selected from the group consisting of N6 -[(3-halothien-2-yl)methyl]adenosine, N6 -[(4-halothien-2- yl)methyl]adenosine, and N6 -[(5-halothien-2-yl)methyl]adenosine.
13. The composition for use of claim 12, wherein the compound is selected from the group consisting of N6 -[(5-iodothien-2-yl)methyl]adenosine, N6 -[(4-iodothien-2- yl)methyl]adenosine, N6 -[(3-iodothien-2-yl)methyl]adenosine, N6- [(5 -bromothien-2- yl)methyl]adenosine, N6 -[(4-bromothien-2-yl)methyl]adenosine, N6 -[(3-bromothien-2- yl)methyl]adenosine, N6 -[(5-chlorothien-2-yl)methyl]adenosine, N6 -[(4-chlorothien-2- yl)methyl]adenosine, and N6 -[(3-chlorothien-2-yl)methyl]adenosine.
14. The composition for use of claim 11, wherein the compound is selected from the group consisting of N6 -[(2-halothien-3-yl)methyl]adenosine, N6 -[(4-halothien-3- yl)methyl]adenosine, and N6 - [(5-halothien-3 -yl)methy 1] adenosine .
15. The composition for use of claim 14, wherein the compound is selected from the group consisting of N6 -[(2-iodothien-3-yl)methyl]adenosine, N6 -[(4-iodothien-3- yl)methyl]adenosine, N6 -[(5-iodothien-3-yl)methyl]adenosine, N6 -[(2-bromothien-3- yl)methyl]adenosine, N6 -[(4-bromothien-3-yl)methyl]adenosine, N6 -[(5-bromothien-3-
yl)methyl] adenosine N76-[(2-chlorothien-3-yl)methy]]adenosine, N6 -[(4-chlorothien-3- yl)methyl]adenosine, and N76'-[(5-chlorothien-3-yl)methyl]ader)osine.
16. The composition for use of claim 11, wherein a therapeutically effective amount of the compound is 0.5-15 mg/kg.
17. The composition for use of claim 11, wherein the composition is administered by an oral, nasal, intravenous, intramuscular, subcutaneous, intraperitoneal or topical route.
18. The composition for use of claim 11, wherein the composition further comprises a pharmaceutically acceptable carrier, excipient or vehicle.
19. The composition for use of claim 11, wherein the subject has insomnia or AD.
20. The composition of claim 19, wherein the insomnia is stress-induced insomnia, caffeine- induced insomnia or a combination thereof.
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| US20160264613A1 (en) * | 2013-10-23 | 2016-09-15 | Academia Sinica | Compounds for use in prevention and treatment of neurodegenerative diseases and pain |
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| US20160264613A1 (en) * | 2013-10-23 | 2016-09-15 | Academia Sinica | Compounds for use in prevention and treatment of neurodegenerative diseases and pain |
Non-Patent Citations (3)
| Title |
|---|
| BYRNE ENDA M., JOHNSON JULIE, MCRAE ALLAN F., NYHOLT DALE R., MEDLAND SARAH E., GEHRMAN PHILIP R., HEATH ANDREW C., MADDEN PAMELA : "A Genome-Wide Association Study of Caffeine-Related Sleep Disturbance: Confirmation of a Role for a Common Variant in the Adenosine Receptor", SLEEP, ALLEN PRESS, LAWRENCE, KS, US, vol. 35, no. 7, 1 July 2012 (2012-07-01), US , pages 967 - 975, XP093194210, ISSN: 0161-8105, DOI: 10.5665/sleep.1962 * |
| HUANG ET AL.: "Adenosine A2A, but not A1, receptors mediate the arousal effect of caffeine", NATURE NEUROSCIENCE, vol. 8, no. 7, 2005, pages 858 - 859, XP055649206, DOI: 10.1038/nn1491 * |
| KUSACHI ET AL.: "Dog Coronary Artery Adenosine Receptor: Structure of the JV6-Aryl Subregion", J. MED. CHEM., vol. 29, 1986, pages 989 - 996, XP001147840, DOI: 10.1021/jm00156a016 * |
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