Increased seizure sensitivity, emotional defects and cognitive impairment in PHD finger protein 24 (Phf24)-null rats.

Phf24 is known as Gαi-interacting protein (GINIP) and is associated with the GABAB receptor. To study the function of Phf24 protein in the central nervous system (CNS), we have newly developed Phf24-null rats and investigated their behavioral phenotypes, especially changes in seizure sensitivity, emotional responses and cognitive functions. Phf24-null rats did not exhibit any spontaneous seizures. However, they showed a higher sensitivity to pentylenetetrazol (PTZ)- or pilocarpine-induced convulsive seizures. Phf24-null rats also showed an elevated susceptibility to kindling development with repeated PTZ treatments, suggesting that Phf24 acts as an inhibitory modulator in epileptogenesis. Although young Phf24-null rats showed normal gross behaviors, elevated spontaneous locomotor activity, especially in terms of the circadian dark period, emotional hyper-reactivity, reduced anxiety behaviors in the elevated plus-maze (EPM) test, and cognitive deficits in the Morris water maze test were explicitly observed at older age (20-week-old). The present results suggest that Phf24 is essential for proper functioning of the CNS, especially in preventing epileptogenesis and controlling emotional and cognitive functions.

Pharmacological characterization of M-II, the major human metabolite of ramelteon.

The duration of action of melatonin may be important for improvements in sleep efficiency in insomniacs. Ramelteon, a selective melatonin agonist, is primarily metabolized to the active metabolite M-II, which has a longer half-life and greater systemic exposure than ramelteon. Hence, M-II may contribute significantly to the hypnotic benefits of ramelteon. We assessed the ramelteon-like activity of M-II in vitro and in vivo using cats. Binding and functional studies in Chinese hamster ovary cells expressing human melatonin receptors (MT1 or MT2) revealed that M-II binds melatonin receptors with lower affinity (Ki: 114 and 566 pmol/l for MT1 and MT2, respectively) and has lower potency (IC50: 208 and 1,470 pmol/l for MT1 and MT2, respectively) compared with ramelteon. However, higher M-II doses significantly improved sleep in cats. Thus, M-II may contribute to the clinical efficacy of ramelteon.

Ramelteon (TAK-375) accelerates reentrainment of circadian rhythm after a phase advance of the light-dark cycle in rats.

In vivo pharmacological effects of ramelteon (TAK-375), a novel, highly MT1/MT2-selective receptor agonist, were studied in rats to determine ramelteon's ability to reentrain the circadian rhythm after an abrupt phase advance. Experiments were also conducted to assess the potential cognitive side effects of ramelteon and its potential to become a drug of abuse. After an abrupt 8-h phase shift, ramelteon (0.1 and 1 mg/kg, p.o.) and melatonin (10 mg/kg, p.o.) accelerated reentrainment of running wheel activity rhythm to the new lightdark cycle. Ramelteon (3-30 mg/kg, p.o.) and melatonin (10-100 mg/kg, p.o.) did not affect learning or memory in rats tested by the water maze task and the delayed match to position task, although diazepam and triazolam impaired both of the tasks. Neither ramelteon (3-30 mg/kg, p.o.) nor melatonin (10-100 mg/kg, p.o.) demonstrated a rewarding property in the conditioned place-preference test, implying that MT1/MT2 receptor agonists have no abuse potential. In contrast, benzodiazepines and morphine showed rewarding properties in this test. The authors' results suggest that ramelteon may be useful for treatment of circadian rhythm sleep disorders without adverse effects typically associated with benzodiazepine use, such as learning and memory impairment, and drug dependence.

The sleep-promoting action of ramelteon (TAK-375) in freely moving cats.

INTRODUCTION: Ramelteon (TAK-375) is an MT1/MT2 receptor agonist being studied for the treatment of insomnia and circadian rhythm sleep disorders. We compared the behavioral effects of ramelteon and exogenous melatonin in freely moving cats. METHODS: Ramelteon and melatonin were each suspended in a 0.5% (weight per volume) methylcellulose solution and administered orally to freely moving cats. In the control trial, each cat was given vehicle. Each dose of ramelteon or melatonin was compared with the vehicle control in a crossover design. Electroencephalogram, electromyogram, and electrooculogram recordings were assessed. RESULTS: Ramelteon significantly decreased wakefulness at doses of 0.001,0.01, and 0.1 mg/kg, increased slow-wave sleep at doses of 0.001, 0.01, and 0.1 mg/kg, and increased rapid eye movement sleep at a dose of 0.1 mg/kg, compared with the vehicle controls, as assessed by analysis of variance. The effects of ramelteon lasted for up to 6 hours when evaluated by reduction of wakefulness. Exogenous melatonin (0.01-1 mg/kg) significantly increased slow-wave sleep, but the effect was weaker than that of ramelteon and lasted for only 2 hours. The lowest doses of ramelteon (0.0001 mg/kg) and melatonin (0.001 mg/kg) had no significant effect on sleep-wakefulness stage. CONCLUSIONS: Ramelteon was more effective than exogenous melatonin in promoting and maintaining sleep in freely moving cats. Based on its unique mechanism of action, ramelteon should be studied further to evaluate its potential for the treatment of sleep disorders.

Effects of ramelteon (TAK-375) on nocturnal sleep in freely moving monkeys.

We investigated the effects of (S)-N-[2-(1,6,7,8-tetrahydro-2H-indeno-[5,4]furan-8-yl)ethyl]propionamide (ramelteon, TAK-375), a novel MT1/MT2 receptor agonist, on nocturnal sleep in freely moving monkeys and compared these results with those of melatonin and zolpidem. Treatment with ramelteon (0.03 and 0.3 mg/kg, p.o.) significantly shortened latency to sleep onset and significantly increased total duration of sleep. Treatment with melatonin (0.3, 1, and 3 mg/kg, p.o.) also decreased sleep latency, but the effect was weak; the only significant reduction was seen with the 0.3 mg/kg dose on latency to light sleep. Melatonin had no significant effects on the duration of sleep. Zolpidem had no significant effects on latency to sleep onset in this study at any dose (1, 3, 10, and 30 mg/kg, p.o.). The highest dose (30 mg/kg) of zolpidem had a tendency to increase slow wave sleep; however, it also induced apparent sedation and myorelaxation. Treatment with ramelteon and melatonin had no evident effect on the general behavior of the monkeys. Spectral analysis (fast Fourier transform, FFT) of both ramelteon and melatonin revealed sleep patterns that were indistinguishable from those of naturally occurring sleep. The EEG power spectra of zolpidem were qualitatively different from that of naturally occurring physiological sleep. Results of the present study support the investigation of ramelteon as a sleep-promoting agent in humans.

Synthesis of a novel series of tricyclic indan derivatives as melatonin receptor agonists.

To develop a new therapeutic agent for sleep disorders, we synthesized a novel series of tricyclic indan derivatives and evaluated them for their binding affinity to melatonin receptors. In our previous paper, we proposed a conformation of the methoxy group favorable for the binding of the MT(1) receptor. To fix the methoxy group in an active conformation, we decided to synthesize conformationally restricted tricyclic indan analogues with the oxygen atom in the 6-position incorporated into a furan, 1,3-dioxane, oxazole, pyran, morpholine, or 1,4-dioxane ring system. Among these compounds, indeno[5,4-b]furan analogues were found to be the most potent and selective MT(1) receptor ligands and to have superior metabolic stability. The optimization of substituents led to (S)-(-)-22b, which showed very strong affinity for human MT(1) (K(i) = 0.014 nM), but no significant affinity for hamster MT(3)() (K(i) = 2600 nM) or other neurotransmitter receptors. The pharmacological effects of (S)-(-)-22b were studied in experimental animals, and it was found that a dose of 0.1 mg/kg, po promoted a sleep in freely moving cats, as demonstrated by a decrease in wakefulness and increases in slow wave sleep and rapid eye movement sleep, which lasted for 6 h after administration. Melatonin (1 mg/kg, po) also had a sleep-promoting effect, though it lasted only 2 h. A new chiral method for the synthesis of (S)-(-)-22b starting from 60, which was prepared from 59 employing asymmetric hydrogenation with the (S)-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl-Ru complex, was developed. (S)-(-)-22b (TAK-375) is currently under clinical trial for the treatment of insomnia and circadian rhythm disorders.