Circadian rhythms are endogenously generated oscillations in behaviour or physiology that occur within a
period of 24 hours (Williams et al.,
2016). Circadian rhythm dysregulation is associated with clinical
manifestation and pathogenesis in psychiatric disorders(Jones and Benca,
2015). Melatonin, the hormone of
the pineal gland is essential for regulating sleep and the circadian rhythm.
The secretion is with a diurnal pattern, increasing soon after the onset of
darkness, peaks in the middle of the night, and gradually falls during the
second half of the night (Brzezinski, 1997). Melatonin has a sedative effect, which could be due to a direct
phase-shifting effect on the suprachiasmatic nucleus, the master clock
controlling circadian rhythms (Arendt and Skene,
2005). Dysregulation of melatonin secretion may contribute to the higher
risk of metabolic disease and early death seen in patients with psychiatric
disease (Nordentoft et al.,
2013). Circadian rhythms disturbances namely insomnia and sleep
disorders are increasingly recognized as a significant factor associated with
poor outcomes of depression and are one
of the most prevalent residual symptoms (Chan et al., 2014; Rethorst et al.,

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Molecular components of the circadian clock

The circadian rhythms are created in a cell-autonomous manner by a transcriptional regulatory network of
circadian clock genes (Kelleher et al.,
2014). This circadian clock controls approximately 10% of all genes (Delaunay and Laudet,
2002; Panda et al., 2002; Ueda et al., 2002).A
cellular transcription-translation feedback loop (TTFL) termed the peripheral
clock is present in each cell governing physiological processes in the tissue-specific way (Lee et al., 2001). The TTFL are involved with a small number of core circadian genes
in which there is the rhythmic production followed by protracted degradation of
protein complexes that negatively impact on their own production  (Young and Kay, 2001). After the identification of the SCN master clock, the first
component of the TTFL was identified as the gene Clock. Consequently, other
“core” members of the TTFL, such as the repressors Period (Per1, Per2, Per3)
and Cryptochrome (Cry1, Cry2, Cry3), activators Arntl (Bmal1) and Npas2, and
nuclear receptors Rev-erb (Nr1d1, Nr1d2) and Ror
(Ror-a, Ror-b, Ror-c) were established (Bhargava et al.,

The basic helix–loop–helix (bHLH)-PAS transcription factors CLOCK
and BMAL1 are positive regulators of the autoregulatory
transcription-translation feedback loops of molecular circadian clocks that are
located in most tissues. CLOCK and BMAL1 heterodimerize and transactivate other
clock genes such as Per1, Per2, Cry1 and Cry2, and downstream clock-controlled
genes via E-box (CACGTG) elements in their promoters (Oishi et al., 2013; Takahashi et al.,
2008b). The
three PER and two CRY proteins, acting as negative elements, enter the nucleus
and assemble into one or more protein complexes (PER complex) interact with CLOCK–BMAL1,
leading to suppression of Clock–Bmal1 transcriptional activity. Regulated
degradation of PER and CRY proteins is followed by re-activation of CLOCK–BMAL1
and the consequent initiation of a new 24-hour transcriptional cycle. Clock
function is further supported by additional negative regulators of Clock–Bmal1,
a coupled transcriptional loop involving nuclear receptors, and
post-transcriptional processes (Kim et al., 2014; Koike et al., 2012; Kume et al., 1999; Sangoram et al.,
1998; Siepka et al., 2007; Solt et al., 2012; Tamayo et al., 2015; Zhao et al., 2007). The
higher values of circadian-clock-related proteins observed at day (BMAL-1 and CLOCK)
and night (PER and CRY) (McClung, 2013). There are a number of other candidate clock components such as
Timeless, Dec1, Dec2 and E4bp4, the roles of which have not yet been clearly
defined (Takahashi et al.,


Clinical relationship among sleep disorder, depression and anxiety

Sleep is governed by the intricate interplay between sleep-wake
homeostasis and circadian rhythms in the body (Breen et al., 2014) and
it is generated and regulated independently from circadian rhythms (Morton, 2013). Breakdown of circadian rhythms is commonplace.
Primary pathophysiology of circadian rhythm sleep disorders is a misalignment
between the endogenous circadian rhythm phase and the desired or socially
required sleep-wake schedule, or dysfunction of the circadian pacemaker and its
afferent/efferent pathways (Mishima, 2013). Primary sleep disorders include those not
attributable to another medical or psychiatric condition: insomnia disorder,
hypersomnolence disorder, narcolepsy, obstructive sleep apnea hypopnea
syndrome, central sleep apnea syndrome, and the parasomnias (Khoury and
Doghramji, 2015). Insomnia is a prevalent sleep disorder, defined by
difficulty falling asleep, difficulty staying asleep, nonrestorative sleep, and
waking symptoms such as fatigue, impaired concentration, and mood disturbance. The
prevalence of insomnia is approximately 5% to 20% in the general adult meeting
stringent diagnostic criteria, and as many as 48% presenting with symptoms (Ohayon, 2002). 

Sleep disturbances are highly prevalent condition and carries significant
burden in terms of functional impairment, health care costs, and are highly interrelated with a depressive status thus included in
the diagnostic criteria for the major
depressive disorder (MDD) (e.g., Diagnostic and Statistical Manual of Mental
Disorders, 5th edition (DSM-V) (Cheung and Yip, 2015).
Clinical, epidemiological and theoretical research has focused on the comorbid
relationship between sleep disturbance and depression (Gold and Sylvia,
2016). To
be diagnosed with major depression, an individual must have at least five of
nine criteria for depressive symptoms, and disordered sleep is one of the nine
symptoms (Tan et al., 2016).
Nearly, 90 % of depressed patients endorse difficulty falling asleep, staying
asleep, and early morning awakenings (Almeida and Pfaff,
2005; Tsuno et al., 2005),
whereas 6 % to 29 % endorse hypersomnia complaints (Roberts et al., 2000).
Several studies have claimed that as high as 70 % of general anxiety disorder
(GAD) patients have complained of sleep disturbance, thereby illustrating sleep
disturbance as an established core symptom of this anxiety disorder (Papadimitriou
and Linkowski, 2005).

Previous research outcomes have suggested that the relationship
between sleep disturbance and depression is bidirectional (Lustberg and
Reynolds, 2000) and that the two conditions could feedback
on each other to mutually maintain their existence (Johnson et al., 2006).
Longitudinal research has demonstrated that sleep disturbance is associated
with an increased risk of developing depression (Baglioni et al.,
2011; Ehlers et al., 1988).
Enormous evidence supports an association between circadian rhythm dysfunction
and mood disorder. (Monteleone
et al., 2011). It
is possible to speculate that a disruption in physiological circadian
rhythmicity characterizes at least a subgroup of depressed patients, thus
opening the way to treatment interventions that, restoring normal endogenous
rhythmic patterns, would resolve depressed states. Therefore, several
pharmacological and non-pharmacological therapies have been developed.

Molecular evidence of circadian rhythm in depression and anxiety

Many lines of evidence in humans as well as in animal models
clearly demonstrate the relationship
between depression and circadian rhythms (Kronfeld-Schor
and Einat, 2012). Chronic
unpredictable stress in rats results in changes in sleep architecture including
in REM sleep patterns in the hippocampus and amygdala (Hegde et al., 2011). In
rats, chronic mild stress results in a reduction
of nocturnal activity level and a flattening of activity rhythm amplitude (Gorka et al., 1996),
development of depression- and anxiety-like behaviours
and a significant reduction in peak
levels of Per2 expression in the SCN (Jiang et al., 2011). An
altered circadian gene expression represents a vulnerability factor associated
with increased risk for depression (Mendlewicz,
The generation of circadian rhythms is instantiated by the transcriptional and
translational feedback loops of several clock genes (Shearman et al.,
Perturbations in one or several of these genes may disrupt the circadian
fluctuation in mRNA and protein expression and possibly also increase the risk for depression (Zheng and Sehgal,
2008). It
has also been documented that circadian-clock-related genes were not
significant in patients with sleep disorders and depressive behaviour due to a flattened or disrupted
rhythmicity of the circadian-clock-related genes (Albrecht, 2013).

Recent research involving depressed patients shows a significant association between TIMELESS and
Per1 (Utge et al., 2010).
With a similar study, significant
associations were found in major depression with SNPs in Cry1 and NPAS2,
whereas significant associations in the bipolar disorder subsample were with
the CLOCK and VIP SNPs (Soria et al., 2010).
Additionally, individuals with a history of depression had higher expression of
CLOCK, Per1, and BMAL mRNA levels (Gouin et al., 2010b).
Several previous research has suggested that melatonin may play a crucial
role in the regulation of
circadian-clock-related gene expression (Johnston et al.,
Unambiguously, melatonin agonist RMT is said to increase the activity of the
MT1 receptors which inhibits arousal signals coming from the suprachiasmatic
nuclei (SCN) that maintain wakefulness and resynchronize SCN and also
stimulates MT2 receptors which synchronize the circadian clock to the day-night
cycle (Norris et al., 2013).
Higher doses of RMT are suggested to produce an improvement in cognition and
neuropathological markers in AD mouse models (McKenna et al., 2012). Although these data from model organisms are
equivocal, the efficacy of RMT as a sleep-promoting agent has been documented
in human subjects in a clinical setting (Roth et al., 2005).

Clock genes in human peripheral blood

Evidence shows that the expression of circadian
clock genes is ubiquitous and that the
majority of tissues throughout the body expresses circadian oscillations in
gene expression (Takahashi et
al., 2008a). Several recent works have shown
that circadian clock genes can affect a wide variety of tissue-specific physiological processes (Ramsey et
al., 2007; Rutter et
al., 2002; Takahashi et
al., 2008a; Wijnen and
Young, 2006). In recent years, several methods have been developed to specifically
access molecular clock components in the periphery. The circadian genes have
been found not only in the SCN, but also in several central and peripheral
tissues such as extra-SCN brain regions, eye, heart, kidney, lung, liver,
skeletal muscle, oral mucosa, and peripheral blood mononuclear cells (PBMCs),
indicating the presence of peripheral circadian oscillators throughout the body
(Buijs and
Kalsbeek, 2001). Among peripheral tissues, the blood is the most accessible and common
tissue source obtainable in clinical settings, circulating leukocytes are ideal
surrogates for studying the human circadian clock system (Hida et al.,
2009a). Interestingly, the clock gene expression profiles in PBMCs are said
to be a useful marker for circadian rhythm in humans (Azama et al.,
2007a). Also, the expression profile of clock genes in human peripheral blood
is said to be affected by age (Hida et al.,

One major difference between PBMCs and peripheral
organs with regard to their synchronisation by the SCN is the absence of neural
control through the autonomic nervous system (Teboul et
al., 2005). It is unclear yet if the peripheral expression of circadian genes
reflects the functioning of the master clock, the SCN of the hypothalamus (Gouin et al.,
2010b). Consistent with the circadian profiles found in other human
peripheral tissues, in human PBMCs, the circadian peaks of Bmal1 and Cry1
expression were phase-advanced and delayed when compared with Per1, Per2,
Rev-erb?, and Dbp (Ebisawa et
al., 2010). Also, an observed segregation in the times of peak HBmal1 and HPer2
expression in PBMCs has been attributed to the existence of molecular
phenotypes that may, in turn, explain the
heterogeneity in clock gene expression (Teboul et
al., 2005).

Chronotherapeutics in circadian rhythm dysfunction and mood disorders

As for pharmacological treatments, several lines of evidence
indicate that antidepressant medications and mood stabilizers influence
circadian rhythms, possibly by acting on serotonin ascending pathways from the
median raphe nuclei, which modulates the sensitivity to light of SCN clock
neurons (Racagni et al., 2007). Chronotherapeutics
approach may correct circadian rhythm abnormalities in many psychiatric
disorders, but in clinical practice, there is still widespread ignorance about
it (Jakovljevic,
2011). Administration
of exogenous melatonin increases sleep quality and length in insomnia patients when endogenous melatonin production
is low (Cardinali et al.,
Melatonin and melatonin receptor agonists have chronobiotic
effects, which mean that they can readjust the circadian system (Quera
Salva et al., 2011). Melatonin
receptor agonists on the market include ramelteon (RMT, Rozerem®), circadin (Circadin®), agomelatine
(Valdoxan®) and tasimelteon (Hetloz®) (Laudon and
Frydman-Marom, 2014).

The U.S. Food and Drug Administration (FDA) has approved RMT, a novel MT1 and MT2 agonist, for treatment of
insomnia in 2004 and by Taiwan Ministry of Health and Welfare in 2014.
Circadin, a prolonged-release formulation of melatonin, was approved by
the European medicines agency (EMA) in 2007 for the treatment of primary
insomnia in patients over the age of 55 years. Agomelatine, a melatonin
receptor agonist with the serotonin receptor (5-HT2C) antagonist
properties, received EMA approval in 2009 for treating depression. Tasimelteon,
a high affinity for both MT1 and MT2, was approved in the U.S. FDA in 2014 for
the treatment of non-24 hour sleep-wake
disorder (Dawoodi et al., 2012; Laudon and
Frydman-Marom, 2014). Although
various drugs are used to treat insomnia, recently synthetic melatonin receptor
agonist, RMT is widely used in the treatment of
insomnia. RMT is a selective MT1 and MT2 receptor agonist having negligible
affinity for other neuronal receptors including gamma-aminobutyric acid and
benzodiazepine receptors complexes located throughout the brain (Liu and Wang, 2012).

Melatonergic agent RMT in circadian rhythm, anxiety, and depression

The melatonin receptor agonists, particularly through activation of
the MT1 receptor, may be effective in attenuating the symptoms, neurochemical
changes, or both associated with the clinical manifestations of depressive
disorders (Liu et al., 2016). The actions of RMT on the MT1 receptor are thought to inhibit the
neuronal firing in the SCN, effectively turning off the arousal signal and
allowing sleep to occur (Zammit et al., 2007). RMT,
similar to melatonin, promotes sleep in cats, nonhuman primates, and humans (Miyamoto et al.,
2004; Roth et al., 2005; Wurtman, 2006; Yukuhiro et al.,
2004) and
phase shifts circadian rhythms in both rodents and humans (Cajochen et al.,
2004; Hirai et al., 2005a; Richardson et al.,
2008). The
longest assessment of both objective and subjective measures in a
placebo-controlled study with the nightly use of RMT in the treatment of chronic insomnia subjects with a 6-month
ramelteon clinical trial reported that the subjects experienced statistically
significantly shorter objective latency to persistent sleep throughout the
6-month study (Neubauer, 2008). Furthermore, RMT was able to treat circadian disturbances without
adverse effects such as learning and memory impairment, and drug dependence in
rats (Hirai et al., 2005b).

The RMT phase advances overt circadian activity rhythms in vivo by
activating the MT1 melatonin receptor, as identical shifts are observed in
wild-type and MT2 knockout C3H/HeN mice (Liu et al., 2016). When treated with RMT, sleep latency and total sleep time
significantly improved compared with baseline and post-study visits in GAD
patients (Gross et al., 2009a).
RMT is especially effective at promoting mood stability in patients who most
recently have had some degree of depressive symptoms and offer neuroprotection
as well (Norris et al., 2013; Srinivasan et al.,
2010). Recently,
in a double-blind, randomized study in patients with the euthymic bipolar disorder and sleep
disturbances, RMT-treated participants were approximately twice as likely to
remain stable throughout the trial as participants treated with placebo. These
facts, suggest the potential benefit of RMT therapy in treating sleep disorders
and its comorbidities.


Aim and Hypothesis

Substantial evidence points out that abnormality in circadian
rhythms might prove causally or pathophysiologically significant in psychiatric
illness. Several studies have reported associations between different circadian
clock genes polymorphisms and mood disorders but no specific clock genes have yet
been reliably related to depression. Based on the fact that the circadian clock
gene expression can be readily measured in PBMCs and displays significant
circadian rhythmicity, making it possible to study central (melatonin levels)
and peripheral (clock gene expression) circadian markers using human blood
samples. Thus, the present study was designed and aimed to investigate the effects
of melatonin receptor agonist RMT on the clinical manifestations of sleep
patterns and the peripheral biological markers of circadian rhythm, including
melatonin levels and the expression profiles of circadian-clock-related genes
in patients with primary insomnia and patients with depression or anxiety with
symptoms of insomnia.

Considering the difficulty in treating patients with insomnia and anxio-depressive symptoms, and based on the treatment
benefits of RMT this present is warranted and relevant. Given the circadian
control of several physiological functions and involvement in psychiatric
disorders, we hypothesized that the disruption in circadian rhythm genes
associated with the insomnia patients suffering from anxiety and depressive
disorders would be restored. Also, we expect that we would find some
significant changes in the peripheral markers which could be used as the
targets for therapeutic interventions in treating patients with insomnia
comorbid with anxiety and depression.

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