Therapeutic potential of melatonin ligands

Melatonin is a neurohormone that is believed to be involved in a wide range of physiological functions. In humans, appropriate clinical trials confirm the efficacy of melatonin or melatoninergic agonists for the MT1 and MT2 receptor subtypes in circadian rhythm sleep disorders only. Nevertheless, preclinical animal model studies relevant to human pathologies involving validated reference compounds lead to other therapeutic possibilities. Among these is a recently developed treatment concept for depression, which has been validated by the clinical efficacy of agomelatine, an agent having both MT1 and MT2 agonist and 5-HT2C antagonist activity. A third melatonin binding site has been purified and characterized as the enzyme quinone reductase 2 (QR2). The physiological role of this enzyme is not yet known. Recent results obtained by different groups suggest: (1) that inhibition of QR2 may lead to "protective" effects and (2) that over-expression of this enzyme may have deleterious effects. The inhibitory effect of melatonin on QR2 observed in vitro may explain the protective effects reported for melatonin in different animal models, such as cardiac or renal ischemia-effects that have been attributed to the controversial antioxidant properties of the hormone. The development of specific ligands for each of these melatonin binding sites is necessary to link physiological and/or therapeutic effects.     

Therapeutic Potential of Melatonin Ligands

Melatonin is a neurohormone that is believed to be involved in a wide range of physiological functions. In humans, appropriate clinical trials confirm the efficacy of melatonin or melatoninergic agonists for the MT₁ and MT₂ receptor subtypes in circadian rhythm sleep disorders only. Nevertheless, preclinical animal model studies relevant to human pathologies involving validated reference compounds lead to other therapeutic possibilities. Among these is a recently developed treatment concept for depression, which has been validated by the clinical efficacy of agomelatine, an agent having both MT₁ and MT₂ agonist and 5‐HT_2C antagonist activity. A third melatonin binding site has been purified and characterized as the enzyme quinone reductase 2 (QR2). The physiological role of this enzyme is not yet known. Recent results obtained by different groups suggest: (1) that inhibition of QR2 may lead to “protective” effects and (2) that over‐expression of this enzyme may have deleterious effects. The inhibitory effect of melatonin on QR2 observed in vitro may explain the protective effects reported for melatonin in different animal models, such as cardiac or renal ischemia—effects that have been attributed to the controversial antioxidant properties of the hormone. The development of specific ligands for each of these melatonin binding sites is necessary to link physiological and/or therapeutic effects.     

Melatoninergic receptor agonists and antagonists: therapeutic perspectives

The chronobiotic neurohormone melatonin, synthetized in the pineal gland during darkness periods governs the circadian and seasonal biological rhythms. Physiologically, melatonin regulates the sleep/activity alternance, together with the circadian cycle of body temperature and cortisol secretion, and influences various immune, endocrine and metabolic functions. Dysfunction of the endogenous melatonin secretion is associated with mood and behavioral disorders including body weight. Patients with severe depression exhibit desynchronized and reduced melatonin secretion, in parallel with marked sleep disturbances whereas exogenous melatonin administration and antidepressive drugs restore melatonin secretion. A dysregulated melatonin secretion is also observed in obese subjects. Implication of melatonin in these disorders stimulated the search for melatonin analogues with enhanced antidepressive and body weight control effects. The melatoninergic agonist S 20098, or agomelatin, disclosed a potent antidepressive and anxiolytic activity in preclinical studies, which was confirmed in clinical trials in patients with major depression. The antagonist S 20928 was shown to limit seasonal weight gain in an hibernating rodent model. Thus, development of melatoninergic agonists and antagonists appear as an innovative approach in the treatment of depression and obesity, two major public health problems.     

Effects of Agomelatine on Oxidative Stress in the Brain of Mice After Chemically Induced Seizures

Agomelatine is a novel antidepressant drug with melatonin receptor agonist and 5-HT(2C) receptor antagonist properties. We analyzed whether agomelatine has antioxidant properties. Antioxidant activity of agomelatine (25, 50, or 75 mg/kg, i.p.) or melatonin (50 mg/kg) was investigated by measuring lipid peroxidation levels, nitrite content, and catalase activities in the prefrontal cortex, striatum, and hippocampus of Swiss mice pentylenetetrazole (PTZ) (85 mg/kg, i.p.), pilocarpine (400 mg/kg, i.p.), picrotoxin (PTX) (7 mg/kg, i.p.), or strychnine (75 mg/kg, i.p.) induced seizure models. In the pilocarpine-induced seizure model, all dosages of agomelatine or melatonin showed a significant decrease in TBARS levels and nitrite content in all brain areas when compared to controls. In the strychnine-induced seizure model, all dosages of agomelatine and melatonin decreased TBARS levels in all brain areas, and agomelatine at low doses (25 or 50 mg/kg) and melatonin decreased nitrite contents, but only agomelatine at 25 or 50 mg/kg showed a significant increase in catalase activity in three brain areas when compared to controls. Neither melatonin nor agomelatine at any dose have shown no antioxidant effects on parameters of oxidative stress produced by PTX- or PTZ-induced seizure models when compared to controls. Our results suggest that agomelatine has antioxidant activity as shown in strychnine- or pilocarpine-induced seizure models.     

Convergence of Melatonin and Serotonin (5-HT) Signaling at MT2/5-HT2C Receptor Heteromers

Inasmuch as the neurohormone melatonin is synthetically derived from serotonin (5-HT), a close interrelationship between both has long been suspected. The present study reveals a hitherto unrecognized cross-talk mediated via physical association of melatonin MT₂ and 5-HT_2C receptors into functional heteromers. This is of particular interest in light of the “synergistic” melatonin agonist/5-HT_2C antagonist profile of the novel antidepressant agomelatine. A suite of co-immunoprecipitation, bioluminescence resonance energy transfer, and pharmacological techniques was exploited to demonstrate formation of functional MT₂ and 5-HT_2C receptor heteromers both in transfected cells and in human cortex and hippocampus. MT₂/5-HT_2C heteromers amplified the 5-HT-mediated G_q/phospholipase C response and triggered melatonin-induced unidirectional transactivation of the 5-HT_2C protomer of MT₂/5-HT_2C heteromers. Pharmacological studies revealed distinct functional properties for agomelatine, which shows “biased signaling.” These observations demonstrate the existence of functionally unique MT₂/5-HT_2C heteromers and suggest that the antidepressant agomelatine has a distinctive profile at these sites potentially involved in its therapeutic effects on major depression and generalized anxiety disorder. Finally, MT₂/5-HT_2C heteromers provide a new strategy for the discovery of novel agents for the treatment of psychiatric disorders.     

Homology modelling of the serotoninergic 5-HT2c receptor

Since its discovery, 5-hydroxytryptamine, more usually called serotonin, has been an elusive candidate as a major mood regulator. This capacity gives it a great importance in the treatment of depression. It is within this framework that our work takes place, as it is related more particularly to a new therapeutic class whose leader is agomelatine. This compound binds to the melatoninergic receptors and to the serotoninergic 5-HT2c receptor, giving rise to the MASSA concept (Melatonin Agonist and Selective Serotonin Antagonist). Like the majority of the serotoninergic receptors, the sub-type 5-HT2c is a G-protein coupled receptor (GPCR). The three-dimensional structure of 5-HT2c is not experimentally known, and we thus resorted to comparative homology modelling to build a model allowing us to study its interactions with agomelatine.     

S20098 AFFECTS THE FREE-RUNNING RHYTHMS OF BODY TEMPERATURE AND ACTIVITY AND DECREASES LIGHT-INDUCED PHASE DELAYS OF CIRCADIAN RHYTHMS OF THE RAT

Mammalian endogenous circadian rhythms are entrained to the environmental day-night cycle by light exposure. Melatonin is involved in this entrainment by signaling the day-night information to the endogenous circadian pacemaker. Furthermore, melatonin is known to affect the circadian rhythm of body temperature directly. A striking property of the endogenous melatonin signal is its synthesis pattern, characterized by long-term elevated melatonin levels throughout the night. In the present study, the influence of prolonged treatment with the melatonin agonist S20098 during the activity phase of free-running rats was examined. This was achieved by giving S20098 in the food. The free-running body temperature and activity rhythms were studied. The present study shows that enhancement of the melatonin signal, using S20098, affected the free-running rhythm by gradual phase advances of the start of the activity phase, consequently causing an increase in length of the activity phase. A well-known feature of circadian rhythms is its time-dependent sensitivity for light. Light pulse exposure of an animal housed under continuous dark conditions can cause a phase shift of the circadian pacemaker. Therefore, in a second experiment, the influence of melatonin receptor stimulation on the sensitivity of the pacemaker to light was examined by giving the melatonin agonist S20098 in the food during 1 day prior to exposure to a 60-min light pulse of 0, 1.5, 15, or 150 lux given at circadian time (CT) 14. S20098 pretreatment caused a diminished lightpulse- induced phase shift when a light pulse of low light intensity (1.5 lux) was given. S20098 treatment via the food was sufficient to exert chronobiotic activity, and S20098 treatment resulting in prolonged overstimulation of melatonin receptors is able to attenuate the effect of light on the circadian timing system. (Chronobiology International, 18(5), 781-799, 2001)     

S20098 AFFECTS THE FREE-RUNNING RHYTHMS OF BODY TEMPERATURE AND ACTIVITY AND DECREASES LIGHT-INDUCED PHASE DELAYS OF CIRCADIAN RHYTHMS OF THE RAT

Mammalian endogenous circadian rhythms are entrained to the environmental day-night cycle by light exposure. Melatonin is involved in this entrainment by signaling the day-night information to the endogenous circadian pacemaker. Furthermore, melatonin is known to affect the circadian rhythm of body temperature directly. A striking property of the endogenous melatonin signal is its synthesis pattern, characterized by long-term elevated melatonin levels throughout the night. In the present study, the influence of prolonged treatment with the melatonin agonist S20098 during the activity phase of free-running rats was examined. This was achieved by giving S20098 in the food. The free-running body temperature and activity rhythms were studied. The present study shows that enhancement of the melatonin signal, using S20098, affected the free-running rhythm by gradual phase advances of the start of the activity phase, consequently causing an increase in length of the activity phase. A well-known feature of circadian rhythms is its time-dependent sensitivity for light. Light pulse exposure of an animal housed under continuous dark conditions can cause a phase shift of the circadian pacemaker. Therefore, in a second experiment, the influence of melatonin receptor stimulation on the sensitivity of the pacemaker to light was examined by giving the melatonin agonist S20098 in the food during 1 day prior to exposure to a 60-min light pulse of 0, 1.5, 15, or 150 lux given at circadian time (CT) 14. S20098 pretreatment caused a diminished lightpulse- induced phase shift when a light pulse of low light intensity (1.5 lux) was given. S20098 treatment via the food was sufficient to exert chronobiotic activity, and S20098 treatment resulting in prolonged overstimulation of melatonin receptors is able to attenuate the effect of light on the circadian timing system. (Chronobiology International, 18(5), 781–799, 2001)     

Agomelatine strongly interacts with zwitterionic DPPC and charged DPPG membranes

Depression is one of the most common psychiatric diseases in the population. Agomelatine is a novel antidepressant drug with melatonin receptor agonistic and serotonin 5-HT_2C antagonistic properties. Furthermore, being a melatonergic drug, agomelatine has the potential of being used in therapeutic applications like melatonin as an antioxidant, anti-inflammatory and antiapoptotic drug. The action mechanism of agomelatine on the membrane structure has not been clarified yet. In the present study, we aimed to investigate the interaction of agomelatine with model membranes of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylgylcerol (DPPG) by Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). We found that agomelatine interacts with the head group in such a manner that it destabilizes the membrane architecture to a large extent. Thus, agomelatine causes alterations in the order, packing and dynamics of the DPPC and DPPG model membranes. Our results suggest that agomelatine strongly interacts with zwitterionic and charged membrane phospholipids. Because lipid structure and dynamics may have influence on the structure of membrane bound proteins and affect the signal transduction systems of membranes, these effects of agomelatine may be important in its action mechanism.     

Major depressive disorder, anhedonia and agomelatine: an open-label study

Despite a wide range of available antidepressants, the effect of the treatment is often suboptimal and there is a need for more effective and better tolerated drugs. Unlike other antidepressants, agomelatine represents a new approach to depression with an innovative mechanism of action. It is an agonist of melatoninergic receptors MT1 and MT2 and a selective antagonist of 5-HT2c receptors. In this open-label 8-week study we aimed to investigate the efficacy of agomelatine on depressive symptoms in patients with major depression. Secondary endpoints were the effect of agomelatine on anhedonia. Thirty major depressive patients received a flexible dose (25-50 mg; per os, daily) of agomelatine. Depressive (Hamilton Depression Scale) and anxious (Hamilton Anxiety Scale) symptoms, anhedonia (Snaith Hamilton Rating Scale), and sleep quality (Leeds Sleep Evaluation Questionnaire) were assessed. Twenty-four patients (80%) completed 8 weeks of treatment. Significant improvements were seen at all visits on the HAM-D (p<.05), HAM-A(p<.01), SHAPS (p<.05), LSEQ (p<.05). Nine subjects (30%) were responders and 5 (17%) remitters at week 1; 18 (60%) were remitters by the end of the trial. There was no serious adverse event. No aminotrasferase elevations were noted. In line with previous studies, in which agomelatine was associated with early clinical improvement, this study also provides evidence of an early response and the findings of improvements in depression scores. Moreover, this is the first study where agomelatine was effective in the treatment of anhedonia. Additional trials are needed to delineate the place of agomelatine in the contemporary pharmacotherapy for depressive disorders.     

Melatonin attenuates the acetylcholine-induced contraction in isolated intestine of a teleost fish

The present study investigates the possible direct actions of melatonin (N-acetyl-5-methoxytryptamine) on intestinal motility in goldfish (Carassius auratus) using an in vitro system of isolated intestine in an organ bath engaged to an isometric transducer. The longitudinal strips from goldfish intestine in the organ bath showed a resting spontaneous myogenic rhythmic activity which is not altered by melatonin. The addition of acetylcholine (1 nmol l⁻¹–10 mmol l⁻¹) to the organ bath induces a significant contraction of the intestinal strips in a concentration-dependent manner. The addition of melatonin and its agonist, 2-iodomelatonin, induced a concentration-dependent attenuation of acetylcholine-induced contractile response. The specificity of this effect is tested by the preincubation of the intestine strips in the presence of two melatoninergic antagonists, luzindole (a non-selective MT₁/MT₂ melatonin receptor antagonist) and 4-P-PDOT (preferred antagonist of MT2 receptor subtype), which counteracted the melatonin-induced relaxation in a concentration-dependent manner. Finally, present results demonstrate that this melatoninergic effect on intestinal strips is a process highly dependent on extracellular calcium. In conclusion, this is the first study demonstrating the role of melatonin in the control of gut motility in a non-mammalian vertebrate. The melatonin effects on isolated intestine from goldfish are mediated by melatoninergic membrane receptors, and could suggest a delay in food transit time, supporting its anorectic effect reported on in vivo studies.     

Design and synthesis of benzoxazole derivatives as novel melatoninergic ligands

A novel series of benzoxazole derivatives was synthesized and evaluated as melatoninergic ligands. The binding affinity of these compounds for human MT(1) and MT(2) receptors was determined using 2-[(125)I]-iodomelatonin as the radioligand. The results of the SAR studies in this series led to the identification of compound 28, which exhibited better MT(1) and MT(2) receptor affinities than melatonin itself. This work also established the benzoxazole nucleus as a melatoninergic pharmacophore, which served as an isosteric replacement to the previously established alkoxyaryl core.     

Disrupted chronobiology of sleep and cytoprotection in obesity: possible therapeutic value of melatonin

From a physiological perspective the sleep-wake cycle can be envisioned as a sequence of three physiological states (wakefulness, non-rapid eye movement, NREM, sleep and REM sleep) which are defined by a particular neuroendocrine-immune profile regulating the metabolic balance, body weight and inflammatory responses. Sleep deprivation and circadian disruption in contemporary "24/7 Society" lead to the predominance of pro-orexic and proinflammatory mechanisms that contribute to a pandemic metabolic syndrome (MS) including obesity, diabetes and atherosclerotic disease. Thus, a successful management of MS may require a drug that besides antagonizing the trigger factors of MS could also correct a disturbed sleep-wake rhythm. This review deals with the analysis of the therapeutic validity of melatonin in MS. Melatonin is an effective chronobiotic agent changing the phase and amplitude of the sleep/wake rhythm and having cytoprotective and immunomodulatory properties useful to prevent a number of MS sequels. Several studies support that melatonin can prevent hyperadiposity in animal models of obesity. Melatonin at a low dose (2-5 mg/day) has been used for improving sleep in patients with insomnia and circadian rhythm sleep disorders. More recently, attention has been focused on the development of potent melatonin analogs with prolonged effects (ramelteon, agomelatine, tasimelteon, TK 301). In clinical trials these analogs were employed in doses considerably higher than those usually employed for melatonin. In view that the relative potencies of the analogs are higher than that of the natural compound, clinical trials employing melatonin doses in the range of 50-100 mg/day are needed to assess its therapeutic value in MS.     

Design and synthesis of 3-phenyltetrahydronaphthalenic derivatives as new selective MT2 melatoninergic ligands. Part II

Following our studies of the melatoninergic receptors, we have developed new tetrahydronaphthalenic derivatives of melatonin that have been tested as selective melatonin receptors ligands. Regarding the role of the phenyl substituent to obtain selective ligands, modulation of selectivity and activity have been achieved by modifications of the acyl group and substitutions on the phenyl ring. Ten of the seventeen evaluated derivatives have MT₂ receptor affinity similar to that of melatonin. Moreover, we have achieved remarkable MT₂ selectivity over MT₁ (selectivity >100) and have been able to further extend the RSA of the tetrahydrophthalenic series. However, the compounds presented here display partial agonist or antagonist behavior instead of full agonist.     

Agomelatine modulates calcium signaling through protein kinase C and phospholipase C-mediated mechanisms in rat sensory neurons

Agomelatine, a novel antidepressant exerting its effects through melatonergic and serotonergic systems, implicated to be effective against pain including neuropathic pain but without any knowledge of mechanism of action. To explore the possible role of agomelatine on nociceptive transmission at the peripheral level, the effects of agomelatine on intracellular calcium ([Ca²⁺]_i) signaling in peripheral neurons were investigated in cultured rat dorsal root ganglion (DRG) neurons. Using the fura-2-based calcium imaging technique, the effects of agomelatine on [Ca²⁺]_i and roles of the second messenger-mediated pathways were assessed. Agomelatine caused [Ca²⁺]_i signaling in a dose-dependent manner when tested at 10 and 100 μM concentration. Luzindole, a selective melatonin receptor antagonist, almost completely blocked the agomelatine-induced calcium signals. The agomelatine-induced calcium transients were also nearly abolished following pretreatment with the 100 ng/ml pertussis toxin, a Gi/o protein inhibitor. The stimulatory effects of agomelatine on [Ca²⁺]_i transients were significantly reduced by applications of phospholipase C (PLC) and protein kinase C (PKC) blockers, 10 μM U73122, and 10 μM chelerythrine chloride, respectively. The obtained results of agomelatine-induced [Ca²⁺]_i signals indicates that peripheral mechanisms are involved in analgesic effects of agomelatine. These mechanisms seems to involve G-protein-coupled receptor activation and PLC and PKC mediated mechanisms.     

Antidepressant effects of combination of sleep deprivation and early evening treatment with melatonin or placebo for winter depression

Patients with winter depression (seasonal affective disorder) respond beneficially to sleep deprivation and bright light, but the mechanisms of these responses remain unknown. The study was designed to test whether afternoon/evening melatonin can prevent further relapse after sleep deprivation (presumably due to a pharmacologically induced advance shift of circadian phase). Compared to phase advancing by alteration of sleep - wake schedule or by bright light exposure, the melatonin intake is a more tolerated treatment procedure, and it provides a possibility of blind comparison between chronotherapeutic and placebo treatments. The depression was scored in 16 female patients with winter depression and 17 age-matched female controls before and after total night sleep deprivation and after subsequent six-day administration of melatonin (0.5 mg) or placebo under double blind conditions. The melatonin intake was scheduled at 17:00 in order to produce a phase advance of circadian rhythms. Sleep deprivation resulted in 38% reduction of depression score in patients, but it did not reduce depression score in controls. After subsequent treatment with placebo or melatonin, slight but significant improvement of mood was found in controls. These treatments also stabilized the antidepressant response to sleep deprivation in patients. However, neither differential effect of melatonin and placebo on depression score nor alteration of habitual sleep timing was found in patients and controls. Thus, the study results do not provide evidence for the antidepressant potential of melatonin in patients with winter depression under realistic clinical conditions. The finding of stabilization of mood in patients with placebo points to the contribution of psychological factors to the therapeutic action of this and other types of innovative treatments for winter depression. To include psychosocial aspects in the theoretical framework of seasonal depression, we conceptualized depression as an evolved feature of emotional response to psychosocial rather than physical environment. The seasonality of depression might be explained by cumulative effects of aperiodical psychosocial factors and periodical physical factors on one of the mechanisms of brain neurotransmission.     

Circadian regulation of bird song, call, and locomotor behavior by pineal melatonin in the zebra finch

As both a photoreceptor and pacemaker in the avian circadian clock system, the pineal gland is crucial for maintaining and synchronizing overt circadian rhythms in processes such as locomotor activity and body temperature through its circadian secretion of the pineal hormone melatonin. In addition to receptor presence in circadian and visual system structures, high-affinity melatonin binding and receptor mRNA are present in the song control system of male oscine passeriform birds. The present study explores the role of pineal melatonin in circadian organization of singing and calling behavior in comparison to locomotor activity under different lighting conditions. Similar to locomotor activity, both singing and calling behavior were regulated on a circadian basis by the central clock system through pineal melatonin, since these behaviors free-ran with a circadian period and since pinealectomy abolished them in constant environmental conditions. Further, rhythmic melatonin administration restored their rhythmicity. However, the rates by which these behaviors became arrhythmic and the rates of their entrainment to rhythmic melatonin administration differed among locomotor activity, singing and calling under constant dim light and constant bright light. Overall, the study demonstrates a role for pineal melatonin in regulating circadian oscillations of avian vocalizations in addition to locomotor activity. It is suggested that these behaviors might be controlled by separable circadian clockworks and that pineal melatonin entrains them all through a circadian clock.     

Synthesis of new N-(arylcyclopropyl)acetamides and N-(arylvinyl)acetamides as conformationally-restricted ligands for melatonin receptors

N-(Arylcyclopropyl)acetamides and N-(arylvinyl)acetamides or methyl ureas have been prepared as constrained analogues of melatonin. The affinity of these new compounds for chicken brain melatonin receptors and recombinant human MT₁ and MT₂ receptors was evaluated using 2-[¹²⁵I]-iodomelatonin as radioligand. Strict ethylenic or cyclopropyl analogues of the commercialized agonist agomelatine (Valdoxan®) were equipotent to agomelatine in binding bioassays. However, the ethylenic analogue was more effective than the cyclopropyl one in the melanophore aggregation bioassay, but was still less potent than the disubstituted 2,7-dimethoxy-naphtalenic compounds.     

Melatonin Signaling Modulates Clock Genes Expression in the Mouse Retina

Previous studies have shown that retinal melatonin plays an important role in the regulation of retinal daily and circadian rhythms. Melatonin exerts its influence by binding to G-protein coupled receptors named melatonin receptor type 1 and type 2 and both receptors are present in the mouse retina. Earlier studies have shown that clock genes are rhythmically expressed in the mouse retina and melatonin signaling may be implicated in the modulation of clock gene expression in this tissue. In this study we determined the daily and circadian expression patterns of Per1, Per2, Bmal1, Dbp, Nampt and c-fos in the retina and in the photoreceptor layer (using laser capture microdissection) in C3H-f+/+ and in melatonin receptors of knockout (MT₁ and MT₂) of the same genetic background using real-time quantitative RT-PCR. Our data indicated that clock and clock-controlled genes are rhythmically expressed in the retina and in the photoreceptor layer. Removal of melatonin signaling significantly affected the pattern of expression in the retina whereas in the photoreceptor layer only the Bmal1 circadian pattern of expression was affected by melatonin signaling removal. In conclusion, our data further support the notion that melatonin signaling may be important for the regulation of clock gene expression in the inner or ganglion cells layer, but not in photoreceptors.