RMT is the favored and well-tolerated treatment choice for chronic insomnia and is suggested that they are associated with improvement in both subjective and objective sleep latency and total sleep time (Kuriyama et al., 2014). Generally, insomnia patients with a comorbid anxiety disorder may respond best to specific hypnotic treatments (Asnis et al., 2015). A recent open-label study showed that RMT treatment reduced sleep latency and an increase in total sleep time, but wake time after sleep onset was not statistically significant with GAD patients with insomnia symptoms (Gross et al., 2009b). A double-blind study, which studied adjunctive RMT treatment in an eight-week trial of outpatients with bipolar I disorder, mild-to-moderate manic symptoms, and sleep disturbance showed that RMT was not superior to placebo in improving insomnia or manic symptoms, but was associated with improvement in a global rating of depressive symptoms which supports our study (McElroy et al., 2011).
Given the data on the sleep rating scores (PSQI) in our study, there were significant differences between the treatment groups. Supporting this, recent studies showed significant improvements in PSQI scores with elderly patients with nocturia and insomnia (Shimizu et al., 2013); in type 2 diabetic patients with insomnia (Tsunoda et al., 2016); and adults with attention-deficit/hyperactivity disorder (Gamble et al., 2013). Thus it is not surprising that RMT showing a potential to treat sleep difficulties and indeed improved the comorbid anxiety and depressive symptoms in our study.
Biological significance of melatonin and melatonin metabolite after chronotherapy
Melatonin is synthesized both in the pineal gland and in a number of peripheral organs and tissues by a process starting with tryptophan conversion to serotonin. Regional distribution of melatonin in different areas of the brain varies and early studies have shown that hypothalamic melatonin concentrations are nearly fifty times higher than in plasma (Pang and Brown, 1983; Srinivasan et al., 2011) While tissue melatonin only exhibits a moderate circadian variation, circulating melatonin exhibits most pronounced circadian rhythm with highest levels occurring at night and very low levels during daytime. Previous studies have described abnormalities of secretion rhythms with advanced phases and/or decrease in nocturnal amplitude of melatonin rhythm in depression (Crasson et al., 2004; Li et al., 2013), even though the nature of melatonin secretion rhythm disturbances in depression remains unclear (Crasson et al., 2004).
Circulating melatonin is metabolized mainly in the liver to form 6-sulfatoxymelatonin (6-HMS), the main metabolite of melatonin in urine (Srinivasan et al., 2011). The urinary 6-HMS could be a good estimate to detect phase-shift in the circadian rhythm of depressive patients compared to control healthy subjects. Our finding shows, urinary 6-HMS concentration was high in patients with insomnia comorbid with anxiety and depression explaining the fact of delayed melatonin production reflecting in urinary 6-HMS which has enhanced instead of lowering in the morning period. This was later recovered similar to the control subjects during treatment period indicating the treatment effect of RMT. In support of our study, a clinical study with the depressed patients showed urinary 6-HMS concentrations higher in the morning compared to night time levels, while these concentrations were lowered from the night to the morning in the control group suggesting that the melatonin production is phase-shifted in major depression (Crasson et al., 2004).
Also, this phenomenon suggests that the secretion of melatonin reached a proper phase shift during the treatment. Also, it may suggest that the RMT treatment may induce an alternative route for melatonin metabolism/utilization. In a study with pre-pubertal children with MDD, showed no difference with the nocturnal excretion of this compound to that of the control group (Waterman et al., 1992). In our study, we observed a clear interaction between a decrease in 6-HMS excretion and an improvement in depressive symptoms after RMT treatment. Low levels of melatonin are usually found in patients with insomnia, particularly elderly patients or those presenting circadian rhythm disorders (Masruha et al., 2010; Zhdanova et al., 2001). Also, similar to our study, the serum melatonin values were not significantly different in ADHD children vs. a control group (Cubero-Millan et al., 2014). This evidence clearly suggest that the phase-shifted melatonin production in insomnia comorbid with anxiety and depressed patients were restored with RMT treatment.
Potentiality of RMT in peripheral circadian clock genes
The rhythmic expressions of clock genes are responsible for circadian rhythms which further govern several aspects of physiology, chiefly at the molecular level. (King and Takahashi, 2000; Panda et al., 2002). The core clock genes build an auto-regulatory feedback loop, including activators (Bmal1 and Clock), repressors (Pers and Crys) and output genes (e.g., Rev-erbs, Dbp and Nfil3) (Roenneberg and Merrow, 2005; Takahashi et al., 2008b). The disruption of circadian genes contributes to various psychiatric disease states (Byrne et al., 2014). Our results provide evidence that abnormalities in clock genes are associated with the psychiatric symptom in terms of levels of expression abnormalities in clock genes especially with clock oscillatory gene Per2 and the expression of the Per2 recovered after the RMT treatment. Per2 generally involves in negative feedback in circadian clock which, along with other clock genes, is essential for the generation of circadian rhythms in mammals. The Per genes being less active, the Per proteins produced less and their amount reaches a maximum at the beginning of the night, and then decreases (Charrier et al., 2017). Several evidences point out that Per1 Per2, and Per3 are found rhythmically expressed in PBMCs (Archer et al., 2008; Azama et al., 2007b; Fukuya et al., 2007; Kusanagi et al., 2008) and are appropriate for estimating an individual’s circadian phenotype (Hida et al., 2009a). Specifically, the presence of a history of depression is mostly associated with higher Clock, Bmal1, and Period1 mRNA expression (Gouin et al., 2010a). The polymorphism of Per2 is related to insomnia and some sleep-related disorders (Li et al., 2015). Recent studies have reported an altered expression of timekeeper genes Per1 and Per2 in cases of shift workers (Husse et al., 2012; Taniyama et al., 2015). Also, supporting our study altered mRNA expressions of Per1, and Per2 in white blood cells in individuals with schizophrenia has been documented (Sun et al., 2016).
Preclinical evidence shows that the Per2 gene appears to be a more functional component of the mammalian circadian clock than the Per1 gene, because functional disruption of Per2 gene causes the complete loss of circadian locomotor activity in mice (Zheng et al., 1999). In a rat model, pretreatment with desipramine, blocked both depressive-like behavior and a decrease in Per2 levels (Jiang et al., 2011). Other findings in rodents subjected to chronic restraint showed a downregulation in Per2 SCN, HC and prefrontal cortex, later restored to control values upon administration of lithium (Kinoshita et al., 2012). In a recent study, which demonstrated the duration-dependent effects of melatonin receptor agonist RMT on clock gene expression in a pancreatic ?-cell line (INS-1 cells) showed a significant decrease in Per1 and Per2 mRNA expression after incubation with ramelteon. The authors also suggested that the decrease in Per1 and Per2 expression were due to the suppression of CREB activity by ramelteon because Per1 and Per2 transcription is activated by CREB (Nishiyama and Hirai, 2014; Travnickova-Bendova et al., 2002). Similarly, the other melatonin receptor agonist agomelatine induced a significant decrease in Per1 and Per2 mRNA in several brain regions restoring of circadian rhythms (Koresh et al., 2012). The role and the mechanism of these variations in Per1 and Per2 need to be further studied. We report that circadian clock gene expression is disturbed in patients with insomnia comorbid anxiety and depression, where the expression of Per2 appears to be particularly altered. Based on our findings, we highlight the importance of instituting regular chronobiological RMT treatment in insomnia comorbid patients thereby strengthening and alleviating circadian rhythmicity to restore life quality for these patients. Moreover, these dysregulated clock genes may be targets for therapeutic interventions in treating patients with insomnia comorbid with anxiety and depression.