Focus Sci. Review Article Feb 2016, Volume 2, Issue 1 Melatonergic Treatment: Focus on Metabolism and Chronobiology Rüdiger Hardeland 1, * 1 Johann Friedrich Blumenbach Institute of Zoology and Anthropology, University of Göttingen, Göttingen, Germany * Corresponding author: Johann Friedrich Blumenbach Institute of Zoology and An- thropology, University of Göttingen, Berliner Str. 28, D-37073 Göttingen, Germany. Tel: +49-551395414. E-mail: [email protected]

Submitted: 01.10.2016 Abstract Accepted: 02.12.2016 Introduction: Melatonin is produced in various organs, but its preferentially nocturnal synthesis and release by the pineal gland is decisive for its chronobiological actions. Keywords: The short half-life of circulating melatonin has been reason for developing synthetic Agomelatine melatonergic agonists. With regard to age- and disease-related dysfunction of the Circadian melatonergic system, treatment with melatonin or its synthetic analogs may be used for Melatonin alleviating health problems with a respective etiology. This review addresses limitations Kynuramine arising from drug-specific metabolism and disregarded chronobiological rules. Ramelteon Metabolism: Differences are illustrated by comparing the metabolism of melatonin and two approved synthetic melatonergic agonists, ramelteon and agomelatine. Apart from © 2016. Focus on Sciences hydroxylation and dealkylation reactions, melatonin can be converted to methoxylated kynuramines, a route absent in the two synthetic drugs. An unsual property is present in the ramelteon metabolite M-II, which still displays melatonergic activity, but attains 30 to 100 times higher levels than the parent compound. Two double-hydroxylated agomelatine metabolites may be involved in sometimes occurring hepatotoxicity. Chronobiology of Melatonergic Drugs: Sleep latency facilitation and readjustment of circadian rhythms require only short actions. Circadian entrainment must consider the phase response curve and requires repetitive well-timed administration. Findings in nocturnally active rodents cannot be generally translated to humans, especially not regarding sleep and, perhaps, not in insulin resistance. Conclusions: Melatonin and synthetic analogs can be suitable for treating sleep-onset difficulties, circadian rhythm sleep disorders and subtypes of depression with an etiology of circadian dysfunction. Applications in the field of metabolic syndrome and insulin resistance have to be seen with caution.

INTRODUCTION secreted by the pineal gland is privileged as a circadian regula- tor. From this gland, the hormone is not only released to the Melatonin (N-acetyl-5-methoxytryptamine) has become circulation, but also, via the pineal recess, in even higher con- known as the hormone of the pineal gland, a role that is asso- centrations directly into the third ventricle of the brain [10]. ciated with the transmission of the signal ‘darkness’ [1], with In mammals, melatonin acts mainly via two membrane-bound, regulation of circadian rhythms at both central and peripheral G protein-coupled receptors, MT1 [11] and MT2 [12], encod- oscillators [2, 3], and, in diurnally active vertebrates including ed by the genes MTNR1A and MTNR1B, respectively. Anoth- the human, with sleep promotion [4, 5]. This is based on a er binding site that was previously believed to represent a third high-amplitude circadian rhythm of melatonin synthesis and melatonin receptor and had been provisionally denominated release with a prominent nocturnal peak. Regulation mecha- as “MT3”, has turned out to be an enzyme, quinone reductase nisms of melatonin biosynthesis have been described in detail 2 (= QR2 = NRH: quinone oxidoreductase 2 = NQO2) [13], [6, 7]. Melatonin is also synthesized in numerous extrapineal for which no signal transduction mechanism has been identi- sites and its total amounts in peripheral organs, especially the fied and which, therefore, cannot be categorized as a receptor. gastrointestinal tract (GIT), exceed the pineal levels by about Regulation mechanisms of signal transduction by MT1 and 400 – 500-fold [2, 7, 8]. This fact is frequently overlooked, but MT2, including receptor interactions with other proteins, and not of particular chronobiological relevance, because extrapi- discussion of further binding sites are reviewed elsewhere neal sites usually contribute poorly to the circulating levels and [14]. Doubts exist concerning retinoid-related orphan recep- exert only minor effects in the circadian system [9]. Except for tors (ROR/RZR), which have been considered as nuclear the retina, amplitudes of extrapineal melatonin rhythms are melatonin receptors and on which a considerable amount of usually low [2, 7]. Thus, in chronobiological terms, melatonin literature exists in the melatonin field. At least, the frequently Rüdiger Hardeland investigated RORα was recently reported to not possess rele- bution within the body. Moreover, with regard to melatonin vant affinity to melatonin [15], contrary to ealier belief. as well as its synthetic analogs, particular attention is neces- Several reasons exist for assuming that the maintenance of sary concerning chronobiological effects, which require a melatonin rhythmicity with high nocturnal levels are import- thorough understanding of the circadian system and its prop- ant to human health [2, 3, 16-18]. In brief, this judgment is erties to warrant a successful application. The aim of this re- based on evidence concerning the decline of melatonin lev- view is to direct the attention to (i) the profound differences els in the course of aging, the even more profound decrease in metabolism of melatonergic agonists, with regard to their observed in several neurodegenerative diseases, the reduction consequences to duration of action, formation of bioactive of melatonin concentrations in numerous other diseases and metabolites and adverse effects, as well as (ii) the necessity disorders, from metabolic syndrome, diabetes type 2, to vari- of understanding fundamental chronobiological issues, such ous neurological and mood disorders, and deviations in mel- as conditions requiring short or long actions, circadian phase atonergic signaling because of polymorphisms of melatonin dependence of efficacy, and limits of translating findings ob- receptors and biosynthetic enzymes. tained in nocturnally active rodents to the human. Melatonergic dysfunction, for whichever of these reasons, has numerous consequences, which are not at all surprising with MELATONERGIC AGONISTS AND THEIR DE­ regard to the pleiotropic, orchestrating role of the pineal hor- VIATIONS IN METABOLISM mone [2]. From a medical point of view, the following areas have become of particular interest: (i) readjustment of devi- An indolic moiety, as present in melatonin (Fig. 1), is not a ating circadian rhythms, in the context of circadian rhythm prerequisite for efficiently binding to 1MT and MT2 recep- sleep disorders (CRSDs) such as delayed sleep phase syn- tors. Only a few clinically studied agonists share this struc- drome (DSPS) and familial advanced sleep phase syndrome tural similarity with melatonin, in particular, TIK-301 (= (FASPS), in mood disorders with an etiology of circadian dys- β-methyl-6-chloromelatonin) and piromelatine [= Neu-P11= function such as forms of seasonal affective disorder (SAD) N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-4-oxo-4H-pyran-2- and bipolar disorder (BP), in blindness and other cases of carboxamide, a melatonin analog in which the N-acetyl group poor entrainment by light/dark cycles or social synchroniz- is replaced by an oxopyran carboxyl structure]. Details and ers; (ii) other forms of depression, however, with limited justi- characteristics of further agonists have been recently summa- fication when aiming treatment at an exclusively melatonergic rized [22]. Various other core structures have been used in the basis; (iii) sleep promotion; (iv) avoidance and counteraction design of melatonergic agonists, such as indoline, indane, te- of metabolic dysregulation, especially with regard to the com- tralin, naphthalene, tetrahydroquinoline, anilinoethylamide, plex of metabolic syndrome, obesity, diabetes type 2 and insu- dihydrobenzofuranyl-cyclopropane and even diphenylamine lin resistance in the brain [4, 5, 16-18]. moieties [22-24]. Although these other structures allow high For purposes of a substitution therapy for individuals with affinities to the membrane-bound melatonin receptors and are decreased pineal function because of age or disease, mela- sometimes of great experimental value in terms of selectivity, tonin has a specific disadvantage. Its half-life in the circulation they are cause of profound differences in agonist metabolism. is remarkably short, usually ranging between 20 and 30 min, This shall be illustrated by comparing the catabolic pathways in the extremes between 10 and 45 min [16, 19]. In princi- of melatonin and two examples of clinically approved drugs, ple, there are two possible solutions to this problem, the use ramelteon and agomelatine (Fig. 1). Melatonin is primarily of controlled-release formulations of melatonin and the de- eliminated by cytochrome P450 (CYP) subforms, mainly via velopment of synthetic melatonergic agonists. Melatonin is 6-hydroxylation, to a smaller extent by demethylation [16]. generally highly tolerable, nontoxic [20] and may become 6-Hydroxymelatonin is conjugated and predominantly excret- critical only under specific conditions such as rheumatic dis- ed as 6-sulfatoxymelatonin. This route is responsible for the eases because of its partially proinflammatory properties [16]. short half-life of melatonin in the circulation. Hydroxylation With regard to effects on the reproductive system, which do at C-atom 6 and other sites can be catalyzed by hydroxyl radi- exist in seasonally breeding mammals, but are considerably cals [25], but 2-hydroxylation is also possible by cytochrome less pronounced in humans, and to assumed developmental c [26]. Another route that is not quantitatively important for effects, administration is cautiously avoided during pregnan- blood levels and excretion, but can become relevant in tissues cy. Up to 300 mg/day of melatonin have been given enterally including the brain, is the kynuric pathway. Numerous reac- to ALS patients for two years without causing problems [21]. tions are known to cleave the pyrrole ring of melatonin, to Nevertheless, only very low doses of 2 mg prolonged-release give N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), melatonin have been approved for patients of 55 years and which may be converted by deformylation to N1-acetyl-5-me- over by the European Medical Agency (EMA). This decision thoxykynuramine (AMK). These reactions include various may appear overcautious with regard to the approval of much enzymatic, pseudoenzymatic, free-radical and photochemical higher doses of synthetic melatonergic agonists (25 or 50 mg reactions as well as dioxygenation by singlet oxygen [27]. In- agomelatine by EMA; 4 or 8 mg of ramelteon by the Feder- terestingly, AFMK seems to be associated with brain inflam- al Drug Administration, FDA) [5, 16]. Numerous synthetic mation [18] and has been reported to exceed by far the CSF agonists have been developed, which are also of interest to concentrations of melatonin, in patients with meningitis [28]. the pharmaceutical industry, since newly invented drugs are Like melatonin [18], AFMK displays neuroprotective prop- patentable. Generally, these compounds exhibit longer half- erties [29], and AMK is a potent scavenger of free oxygen lifes in the circulation and some of them display even higher radicals [30], reactive nitrogen species [31], one of the best affinities to the MT1 and/or MT2 receptors than melatonin. quenchers of singlet oxygen [32], an antiinflammatory agent However, problems can arise from the profound differences and a downregulator of NO formation, as summarized else- in their metabolism and, perhaps, deviations in their distri- where [27]. 2 Rüdiger Hardeland

Figure 1: Melatonin, the synthetic melatonergic agonists ramelteon and agomelatine and examples illustrating several important differenc- es in metabolism. In vertebrates, the main catabolic pathway of melatonin consists of 6-hydroxylation by cytochrome P450 monooxygen- ases, followed by conjugation (mainly sulfation). Other hydroxylation reactions occur under the influence of free radicals. The melatonin metabolite N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) can be formed via numerous enzymatic, pseudoenzymatic and free-rad- ical mechanisms by dioxygenation or double hydroxylation. Metabolites of ramelteon and agomelatine are enzymatically formed, as far as known, by cytochrome P450 enzymes. Deacylation and nitrosation reactions are not included in the figure.

While some similarities in metabolism have to be expect- (Fig. 1). One ramelteon metabolite, usually referred to as ed in other indolic melatonergic agonists, such as TIK- M-II, is generated by this step and displays unusual prop- 301 (except for 6-hydroxylation because of chlorination at erties. Because of its similarity to the parent compound, it this site) and piromelatine [22], the metabolism of struc- is also capable of activating melatonin receptors, with an turally dissimilar agonists has to be entirely different, even activity of about 10% of ramelteon. This is, however, not though the same CYP enzymes can be involved (Fig. 1). In irrelevant, because M-II is substantially longer-lived than ramelteon, the methoxy group of melatonin has been re- the parent compound. Therefore, its concentration in the placed by a dihydrofuran ring, which reduces the CYP-de- blood can attain levels of about 30 – 100 times higher than pendent dealkylation, but which can be cleaved by dioxy- ramelteon [33] and contributes to a remarkably long ac- genation. Since a nitrogen is missing in the 5-membered tion after administration of the drug. Despite the unusu- ring of the indane moiety, a pathway corresponding to the al metabolism, ramelteon does not seem to be associated kynuric route of melatonin is excluded, but in this place a with substantial adverse effects, since it was well tolerated carbonylation site is generated. A further difference con- over periods of about one year [34]. However, it should cerns the replacement of melatonin’s N-acetyl group by an be devoid of that spectrum of actions related to AFMK N-propionyl residue, which may reduce deacylation, but and AMK and, with regard to its chemical structure, it is creates a new site for hydroxylation in the aliphatic chain certainly a poorer direct radical scavenger than melatonin. 3 Rüdiger Hardeland

Profound differences are also apparent in the metabolism promising results on counteracting these metabolic diseas- of agomelatine, in which the indolic moiety is replaced by es [22, 37, 38]. However, the circadian differences between a naphthalene (Fig. 1). A property reminiscent of mela- humans and rodents concerning the phase relation between tonin concerns CYP-catalyzed demethylation, which food and melatonin should be seen as a reason for caution. leads in either case to serotonergic compounds. The re- In fact, recent data have indicated that melatonin may fa- spective agomelatine metabolite S21517 (7-demethylago- vor rather than suppress insulin resistance in humans [39]. melatine) was shown to display binding affinity to 5-HT2C This negative effect of melatonin was reported to be more receptors [35]. As with all naphthalenic compounds, par- serious in carriers of a diabetes type 2-promoting allele of ticular caution is due with regard to oxidotoxicity. Prob- the MTNR1B gene, which encodes the MT2 receptor [40]. lems of this type can arise when double-hydroxylated A further point relevant to melatonergic treatment con- aromates are formed by successive interaction with CYP cerns the distinction between the necessities for melato- enzymes. This is possible when S21517 is hydroxylated at nergic actions of short or long duration. Pharmacologists ring atom 3, or when agomelatine is hydroxylated twice, are usually accustomed to develop drugs and formulations at atoms 3 and 4 (Fig. 1). Compounds of this type may thereof that yield a long persistence in the blood, to assure undergo redox cycling involving naphthoquinone and a sustained availability of the compound. This is desirable naphthosemiquinone intermediates in processes that can for most drugs, but, with regard to melatonin, not generally be driven by ascorbate, as outlined in detail in ref. [36]. for all applications. If the aim is to generate a near-physi- Cases of fulminant hepatotoxicity that have been ob- ological nocturnal pattern, the short half-life of melatonin served with agomelatine, however, only in a limited sub- requires prolonged-release formulations, but these need to population of patients, may be explained by this type of be further improved. To achieve this would be prerequisite redox metabolism [36]. One might speculate on genetic, for substantially supporting sleep maintenance and might drug- or disease-induced differences in the expression be also of value for antioxidative protection. The alternative levels or substrate affinities of CYP subtypes, which might of longer acting synthetic melatonergic drugs has not yet be responsible for the differences in tolerability of agome- sufficed for a relevant support of sleep maintenance [5, 16, latine among patients. For further properties of agomela- 22]. Although statistically significant improvements were tine, especially concerning inhibition of 5-HT2C serotonin reported, the extent of total sleep time extension has rarely receptors, other melatonergic agonists that interfere with exceeded 30 min, whereas several hours would be required serotonin receptors, and their relevance to antidepressant for the needs of insomniacs. effects see refs. [16, 22]. However, sustained actions of melatonin or its synthetic analogs are not generally required. Fast actions of immedi- FUNDAMENTAL ISSUES CONCERNING THE ate-release formulations are fully sufficient for substantially CHRONOBIOLOGY OF MELATONERGIC reducing sleep latency, and this can be achieved by all mel- TREATMENT atonergic agonists tested so-far [5, 16, 22, 23, 41]. In the case of melatonin, relatively low doses, such as 0.1 – 0.3 Disregard of the chronobiological basis of melatonin’s actions mg/day, can already suffice [41], and no convincing neces- can lead, and has led, to misconceptions in both research and sity is obvious for using synthetic agonists at much higher melatonergic treatment strategies. A substantial error may recommended doses. The differences in efficiency between occur in experimental approaches and drug development sleep maintenance and sleep onset can be explained on a if differences between the human as a diurnally active spe- chronobiological basis. Melatonin facilitates sleep onset via cies and the usually nocturnally active laboratory rodents its rapid increase early at night. The action is transmitted are overlooked. In the human, as in other diurnally active by binding to receptors in the circadian master clock, the mammals, high melatonin is associated with physical rest, suprachiasmatic nucleus (SCN), which further acts on the sleep and a pause of food consumption. However, in mice hypothalamic sleep switch, and in the thalamus [5]. In a and rats, high nocturnal melatonin levels are produced in similar way, melatonin can reset circadian rhythms via the a phase of exercise, alertness and food intake. Therefore, SCN, which again only requires short actions, because the studies on sleep promotion in nocturnal rodents, which are strength of phase-shifting signal depends on the relative meaningful with other hypnotics, are without value when change, but not on the sustained absolute value of the mel- using melatonergic agonists, since reductions of alertness atonergic agonist [5, 16, 17]. only indicate suppressive effects in the central nervous When applying short-acting, synchronizing signals, several system rather than soporific actions that reflect physio- additional rules have to be followed. First, resetting signals logical sleep. While it is easy to understand and to accept generally act phase-dependently, i.e., the extent and direc- this difference, which has been, nevertheless, sometimes tion of a phase shift strongly vary within the circadian cy- disregarded, another field of application may be likewise cle. Whether the signal causes advance or delay shifts or re- affected by a comparable misconception. This concerns the mains without effect depends on the time of administration complex of metabolic disorders related to the development and is described by a phase response curve (PRC). In hu- of insulin resistance, such as metabolic syndrome, obesity, mans, the PRC for melatonin is known [42]. To appropri- prediabetic stages, diabetes type 2 and brain insulin resis- ately shift a circadian rhythm, interindividual differences in tance. Food consumption and high melatonin are positively the rhythms of patients must be considered. One possibility correlated in laboratory rodents, but negatively in humans. for determining the individual phase position is to measure Nevertheless, deviations in melatonergic actions have been the evening rise in melatonin concentration, in terms of the described in humans suffering from diabetes type 2 [3, 16, dim light melatonin onset (DLMO) [43]. Moreover, a sin- 17]. Numerous preclinical studies in rodents have yielded gle resetting signal will usually not be sufficient for a stable 4 Rüdiger Hardeland phase shift to a desired temporal position, but will require ncbi.nlm.nih.gov/pubmed/16153306 a series of daily repeated treatments. If clinicians disregard 10. Tricoire H, Locatelli A, Chemineau P, Malpaux B. Melatonin enters the cerebrospinal fluid through the pineal recess. Endocrinology. these basics, they may arrive at an unjustified conclusion on 2002;143:84-90. http://www.ncbi.nlm.nih.gov/pubmed/11751596 inefficiency of the drug or mistake patients with deviating 11. Reppert SM, Weaver DR, Ebisawa T. Cloning and characterization of a circadian period length or atypical phase position for non- mammalian melatonin receptor that mediates reproductive and circa- responders. dian responses. Neuron. 1994;13:1177-85. http://www.ncbi.nlm.nih. gov/pubmed/7946354 12. 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