Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • 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
  • A61K31/7064—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
  • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/20—Hypnotics; Sedatives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

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.
• AD Alzheimer’s disease
• a ⁇ amyloid-beta
• p-tau phosphorylated-tau
• Tau is a protein that stabilizes microtubules in neuronal axons.
• 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-halothien-3- yl)methyl] adenosine, N 6 - [(4-halothien-3-yl)methyl] adenosine, and N 6 -[(5-halothien-3- 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. 6B illustrates the effect of the Entl inhibitor J4 on apoptosis 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. 9 illustrates the oral administration schedule of Ent 1 inhibitor J4 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. 11 illustrates the experimental protocol for the caffeine-induced 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. 13 illustrates the analysis of 24-h NREM sleep alterations after vehicle control (1% HP ⁇ CD) 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% HP ⁇ CD).
• FIG. 14 illustrates the analysis of 24-h NREM sleep alterations after vehicle control (1% HP ⁇ CD) 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% HP ⁇ CD).
• 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.
• FIG. 16 illustrates the analysis of 24-h wakefulness after vehicle control (1% HP ⁇ CD) 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% HP ⁇ CD).
• FIG. 17 illustrates the analysis of 24-h wakefulness after vehicle control (1% HP ⁇ CD) 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% HP ⁇ CD).
• FIG. 18 illustrates the analysis of 24-h wakefulness after vehicle control (1% HP ⁇ CD) 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% HP ⁇ CD).
• FIG. 19 illustrates the analysis of 24-h REM sleep alterations after vehicle control (1% HP ⁇ CD) and oral Entl inhibitor J4 at 1.0 mg/kg.
• FIG. 20 illustrates the analysis of 24-h REM sleep alterations after vehicle control (1% HP ⁇ CD) and oral Entl inhibitor J4 at 6.0 mg/kg.
• FIG. 21 illustrates the analysis of 24-h REM sleep alterations after vehicle control (1% HP ⁇ CD) and oral Entl inhibitor J4 at 12.0 mg/kg.
• 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.
• 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. 24 illustrates the analysis of 24-h REM sleep after oral administering 6.0 mg/kg Entl inhibitor J4 and vehicle control (1 % HP ⁇ CD).
• FIG. 25 illustrates the analysis of 24-h REM sleep after oral administering 12 mg/kg Entl inhibitor J4 and vehicle control (1 % HP ⁇ CD).
• 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% HP ⁇ CD).
• 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% HP ⁇ CD).
• 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).
• 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).
• 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).
• 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).
• 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).
• 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).
• 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. 35 illustrates the alterations of NREM sleep within ZT 1-3 after administration of
• Entl inhibitor J4 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.
• 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)
• FIG. 37 illustrates the effects of cage exchange on REM sleep.
• 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).
• 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).
• 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).
• 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. 43 illustrates the effects of 1.0 mg/kg Entl inhibitor J4 on REM sleep with cage exchange.
• FIG. 44 illustrates the effects of 6.0 mg/kg Entl inhibitor J4 on REM sleep with cage exchange.
• 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).
• 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.
• FIG. 50 illustrates the effects of vehicle and 0.5 % DMSO on NREM sleep.
• FIG. 51 illustrates the effects of vehicle and 0.5 % DMSO on REM sleep.
• 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. 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).
• 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% HP ⁇ CD with icv injection DMSO).
• 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% HP ⁇ CD with icv injection DMSO).
• 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.
• a method of treating schizophrenia 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.
• the compound may be selected from 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 N 6 -[(5-iodothien-2-yl)methyl]adenosine, N 6 -[(4-iodothien-2-yl)methyl] adenosine, M’-[(3-iodothien-2-yl)methyl]adenosine, N 6 -[(5- bromothien-2-yl)methyl]adenosine (also called “JMF3464” or “J4”), N 6 -[(4-bromothien-2- yl)methyl]adenosine, N 6 - [(3 -bromo thien-2-yl)methyl]adenosine, N 6 - [(5-chlorothien-2- yl)methyl]adenosine (also called “JMF3818”), N 6 -[(4-chlorothien-2-yl)methyl]adenosine, N 6 -
• the compound may be selected from N 6 -[(2-halothien-3-yl)methyl] adenosine, N 6 -[(4-halothien-3-yl)methyl] adenosine, and N— 6 [(5-halothien-3- yl)methyl]adenosine.
• the compound is N 6 -[(2-iodothien-3-yl)methyl]adenosine, N 6 -[(4dodothien-3-yl)methyl]adenosine, N 6 -[(5-iodothien-3-yl)methyl)adenosine, N 6 -[(2- bromothien-3-yl)methyl]adenosine, N 6 - [(4-bromothien-3 -yl)methyl] adenosine, N 6 -[(5- bromothien-3 -yl)methyl] adenosine N 6 -[(2-chlorothien-3-yl)methyl]adenosine, N 6 -[(4- chlor othien- 3 -y l)methy 1] adenos ine. or N 6 -[(5-hlorothien-3-yl)methyl]adenosine, or a
• 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.
• a sporadic AD (sAD) mouse is established by the unilateral intrahippocampal (ih) microinjection of A ⁇ aggregates (A ⁇ 1-42 ) 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.
• mice 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.
• 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.
• the connective tissue was wiped with sterilized cotton until the bregma could be clearly seen.
• 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).
• mice 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 24 th 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).
• wakefulness low EEG amplitude and higher frequency
• NREM non-rapid eye movement sleep
• REM rapid eye movement sleep
• mice were randomly divided into four groups: the sham control, vehicle control, STZ+A ⁇ 1-42 , STZ+A ⁇ 1-42 _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+ A ⁇ 1-42 group, mice were infused with icv injection of STZ (3 mg/kg) and ih injection ofA ⁇ 1-42 (1 pg/pl) on day 7 th to 10 th . The ENT1 inhibitor (6 mg/kg) or its vehicle (1% HP ⁇ CD) were administered for 18 days (between days 7 th and 24 th ).
• mice 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).
• OFT open field test
• MLM Morris water maze
• NOR novel object recognition
• 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.
• mice treated with icv-STZ and ih-A ⁇ would induce oxidative stress and subsequently cause DNA damage and neuronal apoptosis as the cholinergic neuronal loss in the MSDB was observed.
• 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 ⁇ M, respectively, obtained from the control and vehicle HP ⁇ CD to 27.82 ⁇ 2.23 pM (p ⁇ 0.05 vs. control or vehicle HP ⁇ CD) in the hippocampus after treated with icv-STZ and ih-A ⁇ .
• the abnormal increase of NO in the hippocampus may further cause DNA damage and cell apoptosis.
• 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-A ⁇ .
• 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 HP ⁇ CD 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).
• 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.
• mice 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).
• Zoletil 10 mg/kg, Carros, France
• 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.
• icv intracerebroventricular
• 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.
• PFS pyrogen-free saline
• 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 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).
• ICELUS M. R. Opp, University of Michigan
• 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
• Example 1 Stress-induced insomnia model
• Example 2 Caffeine-induced insomnia model
• mice 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).
• the experimental mice 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.
• the perfusion needle also known as a butterfly needle
• PFS perfusion fluid
• 4% paraformaldehyde P6148, Sigma- Aldrich, USA
• 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.
• 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.
• O.C.T. Optimal Cutting Temperature
• 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).
• 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.
• PBS Triton X-100
• brain sections were blocked using 3 % normal goat serum (NGS; Jackson Immunoresearch) dissolved in PBS.
• NGS normal goat serum
• slices were stained with the following primary antibody: anti-fos (1.T000; abl90289; abeam) and incubate for 16 hours at 4°C.
• 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).
• Entl inhibitor J4 blocked caffeine-induced insomnia
• 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.
• IP injection of caffeine during the dark-light period produced caffeine-induced insomnia effects.
• FIGs. 26-28 there was a statistically significant decrease during ZT1-2 after oral administration of caffeine when compared to those after administering 1% HP ⁇ CD and PFS.
• 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).
• Entl inhibitor J4 blocked stress-induced insomnia
• 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 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.
• 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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