Binding assays

Cells stably expressing MT1 or MT2 receptors (CHO-hMelR7) were selected and cultured in Eagle’s Minimum Essential Medium-α (MEM-α) supplemented with 10% dialyzed fetal bovine serum (dFBS) under a 5% CO2/95% air atmosphere. Cells were harvested at confluence in Ca2+-Mg2+ free Hanks’ balanced salt solution containing 5 mM EDTA and collected by centrifugation. Cells were homogenized in ice-cold 50 mM Tris-HCl buffer (pH 7.7 at 25℃), washed twice, pelleted, and stored at -30℃ until the binding assays were conducted. Ramelteon and 40 pM 2-[125I]melatonin were mixed with the thawed homogenate in a total volume of 1 mL and incubated at 25℃ for 150 min. The reaction was terminated by addition of 3 mL of icecold buffer followed by vacuum filtration on a Whatman GF/B. The filter was washed twice and radioactivity was counted by a γ-counter. Nonspecific binding was defined as the binding in the presence of 10 mM melatonin.

Cell lines

Pancreatic INS-1 β-cells

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reaction Conditions

2-14 h

Applications

Ramelteon is a potent and selective agonist of MT1/MT2 melatonin receptors. Ramelteon inhibits forskolin-stimulated cAMP production in the CHO cells that express the human MT1 or MT2 receptors [2]. Ramelteon is capable of increasing brain-derived neurotrophic factor (BDNF) protein in neurons expressing either MT1 or MT2 receptor type in mouse cerebellar granule cells [3].

Animal models

Freely moving cats

Dosage form

0.001, 0.01, and 0.1 mg/kg; administered orally.

Preparation method

Suspended in a 0.5% (weight per volume) methylcellulose solution.

Applications

In cats, ramelteon has a sleep-promoting action and does not appear to cause learning, memory, or motor function impairment, or to have rewarding properties [4]. In a clinical study, ramelteon decreases latency to persistent sleep and increases total sleep time and sleep efficiency in subjects with primary chronic insomnia [1].

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

References:

[1]. Kato K, Hirai K, Nishiyama K, et al. Neurochemical properties of ramelteon (TAK-375), a selective MT1/MT2 receptor agonist. Neuropharmacology, 2005, 48(2): 301-310.

[2]. Nishiyama K, Hirai K. The melatonin agonist ramelteon induces duration-dependent clock gene expression through cAMP signaling in pancreatic INS-1 β-cells. PLoS One, 2014, 9(7): e102073.

[3]. Imbesi M, Uz T, Dzitoyeva S, et al. Stimulatory effects of a melatonin receptor agonist, ramelteon, on BDNF in mouse cerebellar granule cells. Neurosci Lett, 2008, 439(1): 34-36.

[4]. Miyamoto M, Nishikawa H, Doken Y, et al. The sleep-promoting action of ramelteon (TAK-375) in freely moving cats. Sleep, 2004, 27(7): 1319-1325.