Application of PBPK modeling for the optimization of oral controlled-release melatonin in ICU patients

Scuola di Ingegneria Industriale e dell’Informazione

Dipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta”

Corso di Laurea Magistrale in Ingegneria Chimica

Application of PBPK modelling for the optimization of oral

controlled-release melatonin in ICU patients

Supervisor: Prof. Davide Manca

Co-supervisor: Eng. Giuseppe Pesenti

Master thesis of:

Mahsa Foroutan Student no. 882905

Parisa Foroutan Student no. 882909

Dedicated to our beloved parents who we take a lot of pride in them and appreciate their encouragement and unconditional love.

Also, to dear respected Dr. Hossein Assarian and Dr. Ali Moradzadeh that we have always received their supersensible supports and great kindness.

Abstract

Sleep deprivation is a major concern for patients in the intensive care unit (ICU) and is usually caused by environmental disturbances, such as high noise levels and continuous lighting, together with increased and intrusive patient care activities. Research has shown that reduced and irregular rhythm of melatonin secretion is one of the most important factors affecting the patients’ sleep conditions during ICU stay. Melatonin is a hormone produced in the body to regulate the daily cycle of sleep and wakefulness and it appears to have the ability to reset the biological clock, thus promoting sleep. Therefore, exogenous administration of melatonin may contribute to the improvement of sleep quality and quantity in ICU patients.

This study aims to find how it is possible to reproduce and restore the healthy concentration profile of endogenous melatonin in ICU patients by the oral administration of exogenous melatonin in a controlled-release form. This means determining the dosage and optimizing the rate of melatonin administration specific to each patient in order to produce a blood concentration trend that resembles the one that naturally arises in healthy people.

In this regard, an existing Physiologically Based Pharmacokinetic (PBPK) model was adapted for oral administration of melatonin to critically ill patients. The model was then identified and validated using experimental datasets from the literature. Although there was a limited amount of experimental data for blood melatonin concentration in ICU patients, our results indicated that the model can fairly predict the average pharmacokinetics of melatonin for a group of ICU patients. Eventually, the PBPK model was applied to investigate the optimal release trajectory and dosage of a controlled-release drug that can mimic the endogenous profile of melatonin in healthy subjects. Results show that sigmoidal functions are the most suitable release trajectories for controlled-release administration of melatonin, leading to a predicted blood concentration profile that is similar to the natural endogenous profile of melatonin.

The PBPK model could be further improved and refined, provided larger experimental datasets are available. We believe that the approach outlined in this work can also be implemented for other special needs of melatonin administration including sleep regulation in shift workers and alleviation

Estratto

La mancanza di sonno rappresenta una grande preoccupazione nei pazienti in terapia intensiva ed è solitamente dovuta a disturbi ambientali, quali elevata rumorosità e luminosità ininterrotta, in combinazione con un’intensificazione di attività invasive per la cura del paziente. Studi hanno dimostrato che un ritmo di secrezione di melatonina ridotto e irregolare è uno dei principali fattori che influenzano i disturbi del sonno durante la permanenza nei reparti di terapia intensiva. La melatonina è un ormone prodotto dal corpo per regolare il ciclo circadiano di veglia e sonno e sembra possedere la capacità di resettare l’orologio biologico, favorendo così il sonno. La somministrazione esogena di melatonina può dunque contribuire ad un miglioramento della qualità e quantità del sonno nei pazienti in terapia intensiva.

Questo studio ha l’obiettivo di stabilire come sia possibile riprodurre e ripristinare il fisiologico profilo endogeno di concentrazione di melatonina nei pazienti in terapia intensiva, mediante la somministrazione orale di melatonina esogena con una formulazione a rilascio controllato. Ciò comporta determinare e ottimizzare la posologia della somministrazione di melatonina specifica per ciascun paziente, al fine di produrre un andamento di concentrazione nel sangue vicino a quello che si verifica naturalmente in persone sane.

A questo proposito, un modello farmacocinetico basato sulla fisiologia (PBPK) già esistente è stato adattato per descrivere la somministrazione orale di melatonina a pazienti in condizioni critiche. Il modello è stato quindi identificato e convalidato impiegando dataset sperimentali presi dalla letteratura. Nonostante la ridotta disponibilità di dati sperimentali di concentrazioni ematiche di melatonina in pazienti in terapia intensiva, i nostri risultati mostrano che il modello è in grado di prevedere correttamente l’andamento farmacocinetico medio di melatonina in un gruppo di pazienti in terapia intensiva. Infine, il modello PBPK è stato applicato per studiare la traiettoria di rilascio e la dose ottimali per una formulazione a rilascio controllato in grado di riprodurre il profilo fisiologico di melatonina. I risultati mostrano che funzioni sigmoidali rappresentano le traiettorie di rilascio più idonee per la somministrazione di melatonina a rilascio controllato, generando nelle simulazioni un profilo di concentrazione ematica che è simile a quello fisiologico.

Il modello PBPK può essere ulteriormente sviluppato e migliorato qualora siano disponibili dataset sperimentali più ampi. Riteniamo che l’approccio presentato in questa tesi possa essere applicato anche ad altri casi particolari in cui sia richiesta la somministrazione di melatonina, inclusa la regolazione del ciclo del sonno dei lavoratori a turni e il lenimento della sindrome da jet-lag nei viaggiatori.

Content index

1.1 RESEARCH CONTENT AND OBJECTIVES ... 11

1.2 MELATONIN ... 14

1.2.1 Melatonin and circadian rhythm ... 15

1.2.2 Benefits and clinical usages ... 18

1.3 SLEEP IN THE INTENSIVE CARE UNIT (ICU) ... 20

1.3.1 Efforts to promote sleep in the ICU ... 26

1.4 PHARMACOKINETICS ... 30

1.4.1 PK modelling ... 34

1.4.2 PBPK modelling ... 36

1.5 ORAL DRUG DELIVERY ... 38

2 METHODOLOGY ... 41

2.1 THE MATHEMATICAL FORMULATION OF THE PBPK MODEL ... 41

2.1.1 Model Parameters ... 52

2.1.2 Model identification ... 57

2.2 PBPK MODEL FOR ICU PATIENTS ... 59

2.2.1 Change of transient times ... 60

2.2.2 ICU model identification ... 61

2.3 CONTROLLED RELEASE IMPLEMENTATION ... 64

2.3.1 Equations of GIT compartments in case of CR administration ... 66

2.3.2 Examples of CR melatonin ... 68

2.4 OPTIMIZATION OF RELEASE TRAJECTORY ... 70

3 RESULTS AND DISCUSSION ... 72

3.1 PBPK MODEL IDENTIFICATION ... 72

3.2 PBPK MODEL VALIDATION ... 74

3.3 COMPARISON OF HEALTHY AND ICU MODELS ... 76

3.4 OPTIMIZED CONTROLLED RELEASE ... 79

3.4.1 Results of CR simulation... 80

NOMENCLATURE ... 90

BIBLIOGRAPHY ... 93

Index of figures

Figure 2. Chemical structure of melatonin (Hardeland et al. 2006). ... 15

Figure 3. Physiology of melatonin secretion (Brzezinski 1997). ... 16

Figure 4. Graphic representation of melatonin biosynthesis (Cardinali et al. 2008). ... 17

Figure 5. Age effect on mean melatonin levels in older people (Scholtens et al. 2016). ... 20

Figure 6. Diurnal variation of endogenous serum melatonin level in 8 critically ill patients in three consecutive days (Olofsson et al. 2004). ... 22

Figure 7. Common factors and their physiological efficacy on sleep deprivation and circadian rhythm disorders in critically ill patients. Light blue circles indicate determinants that are in unidirectional interactions with the possible consequences of sleep loss and circadian rhythm irregularity i.e. green circles. Factors that are in bidirectional interactions, might simultaneously prepare the patients for such disturbances and be their results. Sleep, circadian rhythm disruption, and delirium are in a close relationship (purple circles). Less definite interactions are shown with dashed arrows (Telias and Wilcox 2019). ... 26

Figure 8. Pharmacological interventions to promote sleep in ICU (Hofhuis et al. 2018). ... 27

Figure 9. Serum melatonin levels in healthy subjects on the baseline night and on the simulated ICU noise and light (NL) night (Huang et al. 2015). ... 28

Figure 10. Average serum melatonin concentration profile in four groups of healthy subject in simulated ICU environment, including NL (noise and light), NLEE (wearing ear plug and eye masks in the same noise and light condition), NLP (received placebo in the same environment), and NLM (melatonin was administered under the same ICU environmental condition) (Huang et al. 2015). 29 Figure 11. Representation of the area under the curve (AUC) that is the colored area under the plasma concentration-time profile after oral administration of a drug. AUC is directly proportional to the total amount of unchanged drug that reaches the systemic circulation (Porter and Kaplan 2011). ... 32

Figure 12. Comparison of drug released from conventional release system (via injection or combination of multiple oral dosing) and controlled-release system. The resulting blood levels in plasma are shown by blue dashed curve and red continuous curve, respectively (Huynh and Lee 2015). ... 39 Figure 13. PBPK model structure, the model compartments (single and lumped organs/tissues) are shown by the rectangular boxes including gastric lumen (GL), small intestinal lumen (SIL), large intestinal lumen (LIL),Liver , gastrointestinal circulatory system (GICS), Plasma, salivary glands (SG), pineal gland (PG), poorly perfused tissues (PT) and highly perfused organs (HO). Blue arrows indicate the connections between the compartments which complies with basic physiology of organism, the dashed arrows represent the elimination/metabolism pathways and the black arrows show the possible routes of administration e.g. per os (PO) or transdermal (TD), Savoca et al. (2018). ... 43 Figure 14. Schematic representation of mathematical formulation of SIL’s sub-compartments (Eq. 4-6) ... 45 Figure 15. Reduced parameter model structure, main elimination/excretion pathways shown by circles. Proposed by Savoca et al. (2018) ... 52 Figure 16. Plasma concentration profile after oral administration of 10 mg melatonin. The dots represent mean values and bars represent standard error of the mean (SEM) (Andersen et al. 2016). ... 58 Figure 17. Serum (black circles) and saliva (white circles) melatonin level after administration of 100 mg exogenous melatonin (Vakkuri et al. 1985). ... 58 Figure 18. General conceptual arrangement of the PBPK model (Abbiati et al. 2016). ... 59 Figure 19. Serum melatonin concentration profile of 6 critically ill patients, dashed-red line represents the mean serum concentration (Mistraletti et al. 2010)... 62 Figure 20. Melatonin blood concentration over time for patients received exogenous oral melatonin and placebo (Bellapart et al. 2016). ... 63 Figure 21. Blood melatonin concentration profile before (day 1: endogenous melatonin) and after (day 2) administration of 3 mg melatonin via different routes. The points represent the median values for three groups of seven ICU patients (Mistraletti et al. 2019). ... 64 Figure 22. Linear percentage release profile for three different dissolution times, 40, 80 and 120 min. ... 65 Figure 23. Effect of drug dissolution time, in case of zero-order release (Eq. 26), on the concentration

Figure 24. Comparative percentage release vs. time of Circadin® and melatonin formulations 1, 2, 3, 10, 11 and 12 (Vlachou et al. 2018). ... 68 Figure 25. Comparative percentage release vs. time of Circadin® and melatonin formulations 1 to 9 (Vlachou et al. 2018). ... 69 Figure 26. Schematic diagram of dual drug-loaded hydroxypropyl methylcellulose (HPMC) matrix tablet (Lee et al. 1999). ... 69 Figure 27. Effect of coating levels at two different outer drug concentrations (0.25 and 0.5%) on the release characteristics of melatonin tablet (Lee et al. 1999). ... 70 Figure 28. Endogenous serum melatonin profile of healthy young volunteers, obtained from (Zhdanova et al. 1998). ... 71 Figure 29. Healthy model Identification. Experimental data from Vakkuri et al. (1985). ... 72 Figure 30. Comparison of plasma concentration profiles: in the case of 6 mg single administration of melatonin (red curve), and the case of multiple administration, starting with 3 mg followed by six subsequent 0.5 mg doses hourly (blue curve), according to the study of Bellapart et al. (2016). ... 73 Figure 31. ICU model identification. Experimental data from Bellapart et al. (2016). ... 73 Figure 32. ICU model identification. Experimental data from Mistraletti et al. (2019). ... 74 Figure 33.Comparison between model simulation curve obtained with optimal adaptive parameters and the experimental plasma concentration data obtained after melatonin oral administration for two different dosages (Demuro et al. 2000)... 75 Figure 34. PBPK model prediction (blue solid line) for patients involved in the experimental study of Mistraletti et al. (2010) (red points and SD bars). ... 76 Figure 35. Model predicted plasma concentration curves for two in-silico patients. Red curve indicates the response of model when the in-silico patient is a healthy subject, while blue curve simulates the response of an ICU patient. ... 77 Figure 36. Cumulative liver and total elimination curves for two in-silico patients. Red curve indicates the response of model when the in-silico patient is a healthy subject, while blue curve simulates the response of an ICU patient. ... 78 Figure 37. Initial guess and optimized sigmoidal release curve for a controlled-release melatonin formulation. ... 79 Figure 38. Drug dissolution profile of controlled-release form vs. time. Time 0 is the time of administration which is suggested to be at 5 p.m. ... 80

Figure 39. Comparison of endogenous melatonin profile (Zhdanova et al. 1998) and simulated

plasma concentration vs. time curve, for the in-silico ICU patient. ... 81

Figure 40. Optimized release trajectories for gastric emptying of 120, 280 and 480 min. ... 82

Figure 41. Experimental in vitro data (Vlachou et al. 2018) and the analytical fitting of Circadin® release trajectory. ... 83

Figure 42. Comparison between the model simulation after administration of Circadin® _{with the total} commercial dose (2mg) , the optimal dosage and release trajectory (dotted black line) and the endogenous plasma concentration profile (Zhdanova et al. 1998). The response of the model by implementing in vitro experimental data and the corresponding analytical release trajectory are shown with magenta and blue lines, respectively. ... 83

Figure 43. Comparison between the model simulation after administration of Circadin® _{with 5% of} the commercial dose (100 mcg), the optimal dosage and release trajectory and the endogenous plasma concentration profile (Zhdanova et al. 1998). The response of the model by implementing in vitro experimental data and the corresponding analytical release trajectory are shown with magenta and blue lines, respectively. ... 84

Figure 44. Comparison between optimized release trajectories, orange, red, and dark red lines for gastric emptying time of 120, 280 and 480 min respectively, and in vitro release profile of the commercial drug (Circadin®) as well as the experimental data (light blue lines) for 4 different drug formulations reported by Lee et al. (1999). ... 85

Figure 45. Comparison between optimized release trajectories, orange, red and dark red lines for gastric emptying time of 120, 280 and 480 min, respectively and the experimental in vitro release profiles (blue lines) for 10 different drug formulations reported by Vlachou et al. (2018). ... 86

Index of Tables

Table 2. Body mass fraction and density of different organs and tissues (Brown et al. 1997). ... 54

Table 3. Individualized parameters of the model. ... 56

Table 6. Characteristics of in-silico patient. ... 76 Table 7. Initial guess and optimized values of parameters. ... 79 Table 8. Optimal dosage, time of administration and parameters of release curve for three different GL transit times (120, 280 and 480 min). ... 81

1 Introduction

1.1 Research content and objectives

Since the fifties of last century, on account of advances in biochemistry, molecular and cellular biology, and medicinal chemistry, the process of discovery and development of new drugs has evolved from an empirical approach (i.e. based on empirical evidence from the natural world) to a more hypothesis-driven and mechanism-based approach.

This approach involves the extraction of data from human epidemiology studies, combined with non-clinical in vitro and in vivo experiments, in order to demonstrate the validity of a therapeutic target (Leil and Bertz 2014). Despite the approval rate of new drugs by FDA (the US Food and Drug Administration) has been increasing since 1960, the productivity and efficiency of the traditional approach to drug discovery and development are diminishing (Scannell et al. 2012). This is due to the difficulty of finding new therapeutic targets together with the increase in the costs associated with the discovery and development of new drugs. In order to tackle this negative effect, it is necessary to find viable strategies to simultaneously increase the probability of commercial success of new drugs and decrease the development costs.

In fact, computer-aided modelling is a promising solution for drug discovery, since it allows to explore many experiments in silico to investigate the effect of the drug. Another advantage is that it enables to predict the effect of multiple therapeutic interventions in combination, whereas their testing in clinical studies is usually not economical or even feasible. Thus, computer-aided modeling enables higher commercial successes in drug production and development procedure.

In addition, utilization of computer-aided modelling and simulation increases our understanding of the pharmacokinetics (PK) and pharmacodynamics (PD) of drugs and assists the design of preclinical and clinical experiments.

In general, pharmacokinetics is the study of how an organism (e.g. human body) affects a drug, whereas pharmacodynamics is the study of how the drug affects the organism. The study of both effects together (as in combined PK/PD models) offer possibilities of determining proper dosing, benefits and adverse effects of the administered drug.

Pharmacokinetic models enable the prediction of the drug concentration dynamics in the human body, particularly in the blood. A more detailed explanation about PK models is provided in Subsection 1.4.1. One special branch of pharmacokinetic modelling that focuses on reconciliation of

Pharmacokinetic (PBPK) models. Such models consist of several compartments that correspond to anatomic parts of the body, i.e. specific organs and tissues. Thus, through PBPK models, it is possible to predict drug concentration in different organs and tissues. Further details on PBPK modeling are presented in Subsection 1.4.2.

This work focuses on sleep problems in the critically ill patients, during their stay in the intensive care unit (ICU). Sleep deprivation is a major challenge in critically ill patients and is characterized by poor sleep quality, a paucity of restorative sleep stages and loss of circadian rhythms. It is generally accepted that lack of sleep affects a person's physical and mental health (Lewis et al. 2018; Korompeli et al. 2017). According to the literature, depressed and irregular rhythm of melatonin secretion by the pineal gland, the body's chronological pacemaker, is one of the most important factors affecting the patients’ quality and quantity of sleep during the ICU stay (Perras et al. 2007; Pandi-Perumal et al. 2006).

Melatonin is a chronobiological hormone that plays an important role in the regulation of sleep cycle in humans (i.e., circadian rhythm). Melatonin is secreted by the pineal gland and its production is influenced by the detection of light and dark by the retina of the eyes. Melatonin levels in blood peak during the night-time hours, inducing physiological changes that promote sleep. During the day, melatonin levels are low because large amounts of light are detected by the retinas. Light inhibition of melatonin production is central to stimulating wakefulness in the morning and to maintaining alertness throughout the day (Claustrat et al.2005).

Research has shown that the physiological regulation of melatonin secretion by darkness and light is abolished in critically ill patients (Telias and Wilcox 2019). For this reason, there is a growing interest in the administration of melatonin for sleep regulation in ICU patients. Several studies have investigated the exogenous administration of melatonin in order to reproduce its physiological concentrations. For instance, Zaidan et al. (1994) have studied administration of melatonin via extended intravenous (IV) infusion in healthy volunteers. The results showed that after several, daily-repeated administrations of melatonin, the physiological levels were restored, demonstrating the effectiveness of melatonin as an exogenous synchronizer. Moreover, due to the short half-life of melatonin in the human body, it is believed that a dosage form which can deliver the sustained release melatonin in a circadian fashion would be of clinical value for those who have disordered circadian rhythms (Lee and Min 1996). According to the findings of Mistraletti et al. (2010) and Bellapart et al. (2016), the exogenous oral administration of melatonin in ICU patients is a feasible option with excellent oral bioavailability, however it typically results in supra-physiological plasma

concentration. Therefore, it is essential to find the optimal drug formulation as well as a suitable delivery route and dosage schedule, leading to a plasma concentration profile that mimics the endogenous melatonin concentration profile in the healthy people. Controlled-release administration of melatonin (in more detail discussed in Section 1.5) is a promising solution to achieve this goal.

Apart from this, it has been suggested that exogenous administration of melatonin has several benefits (see Subsection 1.2.1), for instance, it plays an important role as a prophylactic agent in preventing delirium in critically ill patients (Van Den Berghe 2002; Mistraletti et al. 2019).

A search in Scopus of the keywords “exogenous melatonin” revealed 1797 publications between 1963 and 2018. This trend (as illustrated in Figure 1), shows the rising interest in exogenous melatonin studies in the literature.

Figure 1. Rising number of publications about exogenous melatonin, obtained from Scopus.

Given this topic, the objective of this thesis is to (1) develop a PBPK model to investigate the pharmacokinetics of orally administered melatonin in ICU patients and to (2) use this model to optimize the dosage and release trajectory of the drug in order to enhance the sleep condition of critically ill patients. This approach can also be implemented for other special needs of melatonin,

e.g. for sleep regulation in shift workers and for travelers who face the jet-lag syndrome.

The PBPK model implemented in this work builds upon a previous model for melatonin, developed by Savoca et al. (2018) and applied to healthy people. The provided model has been modified in order to make it more suitable for critically ill patients by taking into account their special condition

delayed gastric emptying and lower intestinal motility). The model is trained and validated with experimental data of ICU patients taken from the literature. Subsequently the model is extended in order to describe the administration of controlled-release formulation of melatonin.

Finally, the PBPK model is used for in silico simulations of patients with different physiological characteristics in order to find the optimal dose and release trajectory that can produce a plasma concentration-time profile analogous to the endogenous profile of healthy subjects.

In the following an introduction to melatonin, its benefits and clinical usages and the role of melatonin in the regulation of circadian rhythm is presented. Next the most common sleep problems in the intensive care unit and efforts for improving sleep in ICU are discussed. Finally, the concept of pharmacokinetic study, PK modeling and PBPK modeling are described at the end of Chapter 1. Chapter 2 illustrates the methodology adopted for developing the PBPK model, after which the results are presented and discussed in Chapter 3. Finally, Chapter 4 outlines the main conclusions and makes recommendations for further research.

1.2 Melatonin

The Greek term melatonin literally translates as the tonic of blackness (Pfeffer et al. 2018). Melatonin was discovered by Lerner et al. (1958), who first isolated it from beef pineal gland and named it as a skin-lightening molecule because it produced a lightening effect on the skin color of fish, tadpoles, frogs, and toads, leading them to think it would be useful in dermatology by reversing the skin-darkening effects of melanocyte stimulating hormone (MSH).

Melatonin (N-acetyl-5-methoxytryptamine), a universal photoperiodic hormone, is a small indoleamine with a molecular weight of 232 g/mol (Dikic et al. 2014). This molecule has two functional groups that determine the specificity of its receptor binding and its amphiphilicity (i.e. a

chemical compound possessing both hydrophilic and lipophilic properties), allowing it to enter any cell, compartment or body fluid (Cardinali et al. 2008; Hardeland et al. 2006). Figure 2 shows the chemical structure of melatonin.

Figure 2. Chemical structure of melatonin (Hardeland et al. 2006).

Melatonin is generally known as a physiological hormone released by the vertebrate pineal gland where it is synthesized in a circadian manner with highest values at night, and its secretion is

connected with the regulation of other daily cycles in the body (Reiter and Tan 2002). More details regarding endogenous melatonin secretion in humans are discussed in Subsection 1.2.2.

Aside from night-time synthesis and release of melatonin by the pineal gland, it has been reported that melatonin is also produced in extra-pineal tissues and organs of body, including the retinas, eye lenses, skin, the alimentary canal, bone marrow cells, ovaries, and several blood elements (Dikic et al. 2014; Reiter et al. 2006). It has been reported that high level of melatonin observed in bone marrow cells and bile are probably of non-pineal origin (Reiter et al. 2006). Moreover, melatonin is synthesized by other cells in the body that contain mitochondria. These cells produce melatonin most likely to protect themselves against free radicals (Reiter et al. 2014).

1.2.1 Melatonin and circadian rhythm

Almost all creatures have the ability to anticipate the daily cycle of light and darkness, driven by the rotation of the earth around its axis. The circadian clock (i.e. biological clock) has formed in organisms in order to adjust biological function and processes to a specific time during the day or night. The endogenous master clock which controls several endocrine, physiological and behavioral processes such as the rest/activity patterns, hormonal release, and body temperature, is placed in the hypothalamic suprachiasmatic nucleus (SCN). The physiological clock in the SCN generates an endogenous rhythm of approximately 24 hours, even in the absence of rhythmic environmental cues (Pfeffer et al. 2018).

melatonin) and peptides, and neuroglial cells. In humans and other mammals, the pineal gland acts as a transducer that receives the photic information after the detection of light/darkness by the retina and reflects such information to the suprachiasmatic nucleus (SCN) in the hypothalamus. This neuronal mechanism is initiated by darkness and restrained by light (Brzezinski 1997), as this process leads to the stimulation/suppression of melatonin secretion by the pineal gland, respectively (see Figure 3) (Bedrosian et al. 2013). As mentioned by Haus (2007) melatonin cannot be stored after being released in the pineal gland, so when the synthesis of melatonin in the pineal gland increases, the hormone directly enters the bloodstream through passive diffusion and accordingly the plasma concentrations directly reflect its synthesis and secretion. Moreover, the salivary concentration of melatonin is closely related to its plasmatic concentration at low levels.

Figure 3. Physiology of melatonin secretion (Brzezinski 1997).

In humans, melatonin is synthesized through a series of biological reactions. Firstly, tryptophan (an essential amino acid) is converted to serotonin via 5-hydroxytryptophan. Serotonin is then converted to N-acetylserotonin by the enzyme N-acetyltransferase (NAT). Finally,

N-acetyl-serotonin turns into melatonin by the rate-limiting enzyme, hydroxyindole-O-methyltransferase (HIOMT), as shown in Figure 4 (Cardinali et al. 2008).

Figure 4. Graphic representation of melatonin biosynthesis (Cardinali et al. 2008).

Melatonin can widely distribute in the body thereby applying its actions in almost every cell. Some of these effects are mediated through melatonin receptors, while others are produced independently of receptors. Cell membrane receptors of melatonin are known as MT1 and MT2. Through these receptors melatonin exerts many of its physiological properties, e.g. antioxidant, sleep propensity, control of sleep/wake rhythm, blood pressure regulation, immune function, circadian rhythm regulation, retinal functions, detoxification of free radicals, control of tumour growth, and bone protection (Vlachou et al. 2018; Cardinali et al. 2008)

The human MT2 receptor has a lower affinity with melatonin as compared to the MT1 receptor. Both melatonin receptor subtypes, have been mainly detected in the SCN of most mammals, where they are responsible for chronobiological effects. In vitro and in vivo studies indicated that MT2 receptors mediate the phase-shifting effects of exogenous melatonin (Pfeffer et al. 2018), and MT1 receptors act by suppressing neuronal firing activity (Cardinali et al. 2008).

Melatonin receptors are distributed over a variety of tissues and organs, including, retina, brain, duodenal enterocytes, colon, caecum and appendix, gallbladder epithelium, parotid gland, exocrine pancreas, pancreas, skin, breast epithelium, myometrium, placenta, granulosa and luteal cells, fetal,

immunological actions or to vasomotor control (Cardinali et al. 2008; Srinivasan et al. 2005; Scholtens et al. 2016).

A third melatonin binding site, MT3, was recently characterized as the enzyme quinone reductase. This enzyme belongs to a group of reductases that participate in the protection against oxidative stress by preventing electron transfer reactions of quinones. The MT3 receptor is expressed in the liver, kidney, brain, heart, lung, intestine, muscle and brown adipose tissue, and eyes (Pandi-Perumal et al. 2008).

Onset of melatonin secretion

Melatonin secretion in healthy people increases immediately after the onset of darkness, reaching a peak in the middle of the night (between 2 and 4 a.m.) then slightly reduces until morning (Brzezinski 1997).

In general, the onset time of melatonin secretion from the pineal gland is closely synchronized with the subject's habitual bedtime (occurring approximately 2 hours before bedtime) and is correlated with the onset of evening sleepiness (Cardinali et al. 2008). As outlined by Zhdanova et al. (1998) the evening onset of melatonin secretion, as well as the time of peak concentration, occurred significantly later in a group of younger volunteers than in an older group (by almost 2 hours). Also, the offset of melatonin secretion in the morning also occurred significantly later in the group of younger volunteers than in the older group (again, by almost 2 hours).

1.2.2 Benefits and clinical usages

Numerous benefits of melatonin on the human body have been addressed in the literature. Most commonly, melatonin is regularly employed as a supplement for the treatment of sleep disorders, namely in people with jet lag syndrome and night-shift workers (Brzezinski 1997). Melatonin has been approved for the treatment of primary insomnia in adults older than 55 years, as it can reduce the time to fall asleep and improve the sleep quality and morning alertness in this group of patients. Melatonin was also shown to be effective in treating insomnia in different group of patients, such as children and adults with autism spectrum disorders (ASD), women with premenstrual dysphoric disorder (PMDD) and children with attention-deficit/ hyperactivity disorder (Guénolé et al. 2011; Xie et al. 2017; Hickie and Rogers 2011).

Moreover, as mentioned earlier, ICU patients typically suffer from disruption of the circadian rhythm that is associated with suppressed normal melatonin secretion. Therefore, melatonin as a

sleep regulator can help to improve quality and quantity of sleep in this population. This important application of melatonin, which is the focus of the current work, is discussed separately in Section 1.3.

In addition, melatonin proved to benefit patients suffering from mood disorders, such as seasonal affective disorder and depression. Leppämäki et al. (2003) studied the effect of melatonin on subjects with subsyndromal seasonal affective disorder (s-SAD). According to this study, administration of melatonin significantly improved the quality of sleep and vitality in subjects with s-SAD. Moreover, due to the association of disrupted circadian rhythm and depression, the therapeutic effect of exogenous melatonin on depression and depressive symptoms has been reviewed in several studies (Hansen et al. 2014). For example, Hickie and Rogers (2011) studied the effect of melatonin on major depression and concluded that it produced a significant antidepressant effect through one of its receptor agonist, called agomelatine.

Several experimental studies on human body showed that melatonin inhibits the growth and reproduction of cancer cells (Reiter et al. 2017; Li et al. 2017). Melatonin can prevent proliferation of cancer cells through inhibition of 𝜅B (NF-𝜅B), the transcriptional regulator nuclear factor. Furthermore, the antiproliferative and free radical scavenging action of melatonin might help reducing the gastric tumors prevalence in aged population (Akbulut et al. 2009). The antitumoral effect of melatonin can also be due to the restoration of circadian rhythm. In fact, the circadian rhythm disorders which are hormone-dependent appear to increase cancer risk. Melatonin is also associated in the activation of some immune cells, such as lymphocytes, macrophages and lymphocytes, thus preventing tumor growth in cancer (Zamfir et al. 2014).

Furthermore, anti-inflammatory effects of melatonin were addressed by Reiter et al. (2006). Reportedly, melatonin treatment reduces damage to the tissue during inflammatory reactions thanks to the scavenging of free radicals (i.e. direct effect) and the reduction of the production of agents which contribute to cellular damage (i.e. indirect effect).

Other functions of melatonin include its direct and indirect effects on reproductive systems and gonadal (i.e. sex gland) (Talpur et al. 2018). Melatonin regulates the activity of gonadal according to seasonal changes, meaning that in winter ovulation usually occurs in the evening, but in the summer when the night hours becomes shorter, ovulation usually occurs in the morning (Dikic et al. 2014).

grow and protect it from oxidative damage (Reiter et al. 2014), but also interferes with the process of egg maturation and ovulation (Das Chagas Angelo Mendes Tenorio et al. 2015).

Both in vitro and in vivo studies have shown that melatonin is a potent scavenger of the highly toxic hydroxyl radical and other oxygen-centered radicals and it seems to be more effective than other known antioxidants (e.g. mannitol, glutathione, and vitamin E) in protecting against oxidative damage (Reiter 1995). It should be noted that melatonin requires a higher concentration than its physiological levels to exert its antioxidant effects (Brzezinski 1997).

It is worth noting that melatonin levels changes during the lifetime. Although there is a high inter-individual variability between subjects, nocturnal melatonin levels are highest in young children (approximately 325 pg/ml) and will decrease gradually with age. Figure 5 illustrates this trend even among older people. Generally, it is assumed that higher melatonin levels can play a critical positive role in healthy aging and life expectancy, whereas lower melatonin concentration in older people is associated with disturbances of circadian rhythm, e.g. sleep disorders and delirium (Scholtens et al. 2016). The decrease in nocturnal serum melatonin concentrations (serum is a clear yellowish colored fluid which is part of the blood and does not contain white or red blood cells or clotting factors) that occurs with aging, together with its multiple biologic effects (e.g. protection of brain from oxygen-base radicals and protecting all body cells against oxidative attacks), has led several investigators to suggest that melatonin has a role in ageing and age-related diseases (Reiter 1995).

Figure 5. Age effect on mean melatonin levels in older people (Scholtens et al. 2016).

1.3 Sleep in the Intensive care unit (ICU)

Generally, sleep is managed by two major regulative systems: a circadian system that runs with 24 h periodicity, and a homeostatic system that assure adequate quantities of sleep are obtained. Both

processes are commonly disturbed in critically ill patients due to the contribution of diverse factors (Telias and Wilcox 2019). In fact, sleep disruption in ICU is one of the most frequent complaints of patients. In a study that performed on ICU patients during an 8-months period, sleep disruption was scored as the second most stressful factor (Drouot et al. 2008). However, ICU patients mainly experience sleep deprivation qualitatively, but not necessarily quantitatively. In fact, the total sleep time during a day for the ICU patients is similar to that of a non-hospitalized subject, but there are marked differences in sleep architecture. In general day time naps are common in ICU patients due to loss of light/dark circadian regulator and long inactive decubitus. Moreover, as about half of a critically ill patient’s sleep occurs during daytime hours, the length and frequency of nocturnal sleep are decreased, and frequent awakening lead to high sleep fragmentation in this kind of patients

(Telias and Wilcox 2019; Freedman et al. 2001; Drouot et al. 2008).

Many studies in the literature agree that the intensive care unit environment is not conducive to restorative sleep and outline the reasons behind sleep deprivation and disturbances in the ICU. As presented in the following, these studies have presented the physiological, psychological, and environmental factors as the main contributing factors. However, due to the number and complexity of interactions between these aspects, information regarding the exact mechanisms leading to these disruptions is still lacking and does not allow determining the individual importance of each factor. In the following the main causes of sleep problems in ICU are explained.

Endogenous melatonin secretion

In addition to sleep disturbance, ICU patients more generally feature alterations of their circadian rhythm. The most important evidence for this phenomenon is irregular and suppressed secretion of endogenous melatonin as a robust circadian pacemaker, affecting the patients' quality and quantity of sleep during ICU stay. The findings of Olofsson et al. (2004) clearly demonstrate this behavior. They have studied serum and urine melatonin levels over three consecutive days in eight critically ill patients during deep sedation and mechanical ventilation. Figure 6 shows their endogenous serum melatonin level, which was sampled every 4 hours during the experiment interval. Values are shown in two separate diagrams for the sake of clarity. The curve indicated by “normal” illustrates a hypothetical profile for mean values of melatonin among healthy individuals over the course of three consecutive days. Although melatonin concentration values varied significantly between the studied patients, the melatonin secretion rhythm was disrupted in all but one patient (patient 8,

suggested that administration of melatonin seems to be helpful for treating such irregularity of melatonin secretion.

Figure 6. Diurnal variation of endogenous serum melatonin level in 8 critically ill patients in three consecutive days (Olofsson et al. 2004).

ICU environment

External or environmental factors that are called zeitgebers (literally translated as “time giver”) harmonize biological rhythm in humans with the light/dark 24 h cycle. Consequently, one of the main reasons for disordered normal sleep/wake cycle and aggravation of a pre-existing sleep disorder in ICU patients is the absence of effective zeitgebers in the ICU environment, such as natural lighting. While night-time light levels in ICU are normally less intense than the day-time levels, during some clinical procedures light devices deliver significantly higher light levels up to 100 folds, resulting in a supposed impact on patients’ circadian rhythms. Accordingly, several studies that focused on modulating light exposure have shown that reducing light intensity in critically ills

decreases the occurrence of delirium (i.e. acute confessional state) (Telias and Wilcox 2019; Verceles et al. 2013).

However, the importance of light as a disturbing factor on of patients' sleep and circadian rhythm may vary depending on the light intensity in each intensive care unit (Weinhouse and Schwab 2006). In addition, ICU noise has been highlighted as an effective factor on sleep disturbance in critically ill patients. A reason is that, environmental noise was found to result in release of adrenaline which prevents relaxation and consequently prohibits the patient from falling asleep.

The most trivial contributing sources of sound disruption are noise from equipment such as alarms, monitors, ventilators and other equipment, staff conversation, telephone rings and patient care interventions. Numerous ICU studies have reported average noise level as twice as the maximum sound levels specified by the World Health Organization (WHO). Moreover, diurnal and nocturnal ICU noise levels have been found to be similar as well as the noise level in both sleep and wakefulness periods. Such noise condition leads to lower quality and quantity of sleep in ICU admitted patients (Telias and Wilcox 2019; Tembo and Parker 2009).

Also Freedman et al. (2001) have stated the role of ICU noise on sleep quality, but not necessarily on its quantity, in the ICU. However, they have found that ICU environmental noise is not the major responsible for sleep fragmentation and disruptions. Therefore, noise may not be as disruptive to sleep as other researchers suggested.

Loss of physical activity

Physical activity is another powerful zeitgeber that affects circadian rhythm functionality. In an experimental study in which healthy subjects were requested to remain in bed for 36-h duration, daytime naps were common. Similarly, in astudywhere healthy volunteers stayed in bed in anICU, most of the volunteers lost their circadiansleep and slept during the day. Their findings suggested that the lack of daytime activity itself may be another reason for disrupted circadian rhythms in critically ills (Drouot et al. 2008).

Intrinsic factors and treatments

According to the literature, it is still uncertain whether circadian rhythm irregularity observed in the critically ill patients is a compensatory response to their ICU stay or it comes from their health condition itself. While it is difficult to separate the effects of the illness on circadian rhythms from

the impacts of the ICU environment itself, several researchers outlined the severity of illness as a considerably important factor on sleep disruptions.

In particular, the severity of critical illness results in increased production of catecholamines (a hormone which produce while physically or psychologically being stressed) which leads to sleep disturbance. Also, inflammatory mediators produced during sepsis are also believed to have an impact on altering normal sleep patterns, while non-septic patients in the ICU showed to be relatively able to maintain circadian rhythms comparable with non-hospitalized subjects (Korompeli et al. 2017).

In addition, disease severity may be a major factor in highly fragmented and nonconsolidated sleep, as greater numbers of arousals and awakenings per hour were observed in patients with higher severity scores and in patients who died (Drouot et al. 2008; Tembo and Parker 2009).

Similarly, findings of Gabor et al. (2003) suggest that in the same open-plan ICU environment, critically ill patients slept more poorly than did healthy volunteers, with a higher awakening index, shorter sleep time and more tendency to sleep during the day unlike healthy volunteers.

Apart from acute illnesses itself, sleep may be affected by the resultant pain and discomfort, anxiety, mood disorders, nursing care, and mechanical ventilation (Drouot et al. 2008).

Sleep fragmentation and disturbances in ICU patients under mechanical ventilation are common and depend on the mode of ventilation. In fact, in addition to all the problems known to affect the sleep of non-ventilated patients, mechanically ventilated ICU patients also deal with dyssynchronous breathing, ventilator mode, discomfort from the endotracheal tube and masks, stress related to increased difficulty communicating, and possibly a greater severity of illness. Thus their sleep may be further worsened and they have greater difficulties in sleeping compare to other ICU patients (Tembo and Parker 2009; Weinhouse and Schwab 2006).

In addition, sedatives and analgesics used to promote sleep and comfort in critical care such as benzodiazepines, opioids, inotropes (catecholamines), antihypertensives, antipsychotics, and antidepressants were reported to be among the contributing factors to sleep disorders (Drouot et al. 2008) as well as having other side effects on humans such as depressing the respiratory system function (El-Khatib and Bou-Khalil 2008).

Consequences of sleep problem in ICU

Altered patterns of sleep during an ICU stay take days to normalize and may persist for an extended period after being discharged. Accordingly, ICU patients are associated with several consequences

of sleep deprivation both during and after ICU maintenance, affecting their quality of life (Telias and Wilcox 2019; Weinhouse and Schwab 2006). The complex nature of prolonged sleep disturbances makes them a challenging syndrome in ICU. As mentioned earlier, the underlying reasons are multifactorial, accordingly, they can have several effects in the whole body whose study requires a multi-disciplinary approach. For instance, many researchers addressed the relationship between sleep disorders and functionality of immune system, which is most likely due to the direct effect on the central nervous system (Weinhouse and Schwab 2006). Sleep loss in ICU patients may decrease the strength of immune responses that could hamper weaning from assisted ventilation, prevents the body’s natural defense mechanisms designed to deal with the outcome of injury or illness as well as reducing cognitive capacity and emotional resilience (Tembo and Parker 2009).

Moreover, extended sleep deprivation can result in perceptual distortions and hallucinations in healthy individuals. These effects may play a role in the common occurrence of delirium in ICU patients that lengthens their ICU stay and increases mortality. Although, one study contended that sleep deprivation is actually not the cause of delirium but that it is delirium which causes sleep deprivation (Korompeli et al. 2017), Tembo and Parker (2009) stated that ICU delirium could be both cause and effect of sleep deprivation simultaneously.

In addition, sleep loss has been reported to affect human body’s metabolism, it may cause impairment of normal secretion of hormones and reduce pain tolerance and increase fatigue on sympathetic nerve centers. Moreover, it can lead to irritability, memory loss, inattention, delusions, slurred speech, incoordination, blurred vision, psychological or neurocognitive dysfunction, and

may increase nocturnal high blood pressure (Korompeli et al. 2017; Tembo and Parker 2009; Weinhouse and Schwab 2006).

Figure 7 illustrates the main elements and physiological consequences of sleep and circadian rhythm disruptions in the ICU (Telias and Wilcox 2019).Also, unidirectional and bidirectional interaction between causes and effects of sleep impairment in ICU are presented. Most of the factors are changed by determinants related to the disease consequences such as pain, inflammation and end-organ dysfunction. Moreover, previous sleep disturbances are common in many patients admitted to the ICU, possibly elevate the risk of having them during ICU stay.

Figure 7. Common factors and their physiological efficacy on sleep deprivation and circadian rhythm disorders in critically ill patients. Light blue circles indicate determinants that are in unidirectional interactions with the possible consequences of sleep loss and circadian rhythm irregularity i.e. green circles.

Factors that are in bidirectional interactions, might simultaneously prepare the patients for such disturbances and be their results. Sleep, circadian rhythm disruption, and delirium are in a close relationship (purple circles). Less definite interactions are shown with dashed arrows (Telias and Wilcox

2019).

1.3.1 Efforts to promote sleep in the ICU

Many studies have assessed interventions in the ICU with the aim of improving nocturnal sleep, including non-pharmacologic sleep bundles, light therapy, noise reduction protocols (e.g. use of earplugs), relaxation techniques (e.g. music therapy) and differing modes of mechanical ventilation. However, most of these studies have had limited success (Telias and Wilcox 2019). For instance, Engwall et al. (2015) set up a cycled lighting system to reconstruct the natural light/dark cycle in ICU, in order to promote regulation of the patient’s circadian rhythm. The authors concluded that such intervention engendered feelings of calm and security but could not demonstrate a certain effect on the nocturnal sleep of studied patients. On the contrary, a study on 10 elderly patients admitted to ICU for acute cardiac, respiratory or renal diseases, showed progressive resynchronization of circadian rhythm after decreasing the nocturnal lighting levels. Accordingly, the authors suggested that environmental conditions (light, noise) should be considered to maintain regular rest–activity rhythm in hospitalized subjects (Vinzio et al. 2003). A study by Demoule et al. (2017) showed that the use of earplugs and eye masks at night in ICU patients who have been

awoken from the effects of sedation could decrease the number of prolonged awakenings. Despite this, patients could poorly tolerate wearing these devices and the study was met with limited success, as they are applicable only to patients that are compliant and willing to wear them (Hofhuis et al. 2018).

In addition, pharmacological therapies are also common in ICU in order to improve sleep problems and tackle circadian rhythm irregularities. For instance, sedatives are medications commonly used to promote sleep in ICU, but their administration dose should be minimized if they cannot be avoided since drugs such as opioids, benzodiazepines, and propofol are known to have several side effects. For instance, they affect the normal sleep physiology, reduce the clinician’s ability to monitor the level of consciousness, and induce respiratory depression (Huang et al. 2015). Moreover, although sedatives may increase total sleep time, sleep quality improvements are not guaranteed, i.e. administration of these medications may merely induce a sedative state (Telias and Wilcox 2019).

Although minimal empirical evidence is available to understand the best agent to promote sleep in the ICU, many studies have assessed the efficacy of melatonin as a sleep supplement. A multinational survey study by Hofhuis et al. (2018) was performed in ten European countries to describe sleep assessment and strategies to promote sleep in 522 adult ICUs. Their findings showed that melatonin was the third most common agent used in studied ICUs to promote sleep (Figure 8).

Figure 8. Pharmacological interventions to promote sleep in ICU (Hofhuis et al. 2018).

In addition to the numerous benefits of melatonin that have been discussed in Subsection 1.2.1, administration of exogenous melatonin has been suggested to play an important role in preventing delirium in critically ill patients. Moreover, the pleiotropic function of melatonin in different

physio-pathological processes have been studied in detail and showed to display hypnotic, analgesic, antiseptic and physiological sleep regulatory effect on critically ill patients (Mistraletti et al. 2019). Huang et al. (2015) conducted a study on healthy subject in a simulated intensive care environment in order to evaluate the effect of oral exogenous melatonin as well as using earplugs and eye masks on the nocturnal sleep condition. According to their findings, both night-time sleep and body production of melatonin were disturbed in healthy subjects when they were exposed to simulated ICU noise and light (Figure 9).

Figure 9. Serum melatonin levels in healthy subjects on the baseline night and on the simulated ICU noise and light (NL) night (Huang et al. 2015).

The research was performed on 4 groups of 10 subjects for 8 nights. First, all the subjects were exposed to simulated ICU noise and light for three days, after that, the study was divided into 4 groups. Subjects of the first group were only exposed to ICU noise and light (NL), the second group used ear plug and eye masks in the same condition (NLEE), the third group received placebo (NLP) and the last group was orally administered 1 mg of melatonin (NLM). The results of melatonin concentration measurements after each experiment are illustrated in Figure 10. As can be seen, both oral melatonin and the use of earplugs and eye masks during a simulated ICU environment elevated melatonin levels to some degree. However, the administration of exogenous melatonin was found to be much more effective. Moreover, the discomfort of earplugs and eye masks possibly can affect sleep quality, while no adverse effects of oral melatonin have been reported in this study. Therefore, melatonin has showed better performance in effectiveness and patient tolerability compared to other invention in this study.

Figure 10. Average serum melatonin concentration profile in four groups of healthy subject in simulated ICU environment, including NL (noise and light), NLEE (wearing ear plug and eye masks in the same noise

and light condition), NLP (received placebo in the same environment), and NLM (melatonin was administered under the same ICU environmental condition) (Huang et al. 2015).

Similarly, Bourne et al. (2008) examined the effect of exogenous melatonin on nocturnal sleep in 12 ICU patients being weaned from mechanical ventilation. In this study 10 mg of immediate-release melatonin (i.e. the drug form that releases quickly after administration, see Section 1.5) was administered enterally at 9 p.m. for four consecutive nights. However, pharmacokinetic analyses showed that a 10 mg dose of melatonin leads to supra-physiological morning plasma levels in critical care patients. In order to minimize the risk of daytime high melatonin concentrations, the authors suggested a dose of 1 to 2 mg for future studies to provide suitable nocturnal plasma melatonin concentrations. They concluded that the group of patients who received oral melatonin

demonstrated improved nocturnal sleep efficiency compared to placebo.

Shilo et al. (2000) also studied the effect of exogenous melatonin on the sleep of eight patients in pulmonary ICU. Subjects received 3 mg of controlled-release melatonin or placebo tablets at 10 p.m. on two consecutive nights. Their findings showed that oral melatonin significantly improved quality (reduced sleep fragmentation) and quantity of nocturnal sleep in all the patients that was comparable with general ward patients. Authors enumerated numerous benefits after melatonin administration, such as a possible resynchronization of the circadian rhythm, prevention of ICU syndrome, and acceleration of healing. They concluded that melatonin treatment was safe and effective to improve sleep in ICU, but also state that further studies are needed to assess its efficacy in long term administration.

Moreover, findings of Mistraletti et al. (2010) confirmed that enteral administration of melatonin is a feasible option with excellent oral bioavailability in the critically ill, even though enteral melatonin administration is associated with high first-pass effect and pharmacological interactions on its enteral absorption in ICU patients.

It is important to note that, in order to increase the efficacy of any sleep-inducing medication in ICU, their administration should be accompanied by non-pharmacologic interventions (e.g. daytime mobilization and attempts to keep patients awake during daytime hours).

Finally, most likely a successful sleep improvement plan should consider both internal and external disturbing determinants of sleep in ICU to maximize its efficacy (Telias and Wilcox 2019).

1.4 Pharmacokinetics

Pharmacokinetics (PK) is a branch of pharmacology that studies the fate of substances administered to the human body. The substances of interest can include any natural compound or synthetic chemical, such as pharmaceuticals, food additives, cosmetics, etc.

The pharmacokinetics of a drug describes the different effects that the human body produces on the administered amount. After administration, drug molecules undergo several processes known as ADME (i.e. absorption, distribution, metabolism and excretion).

The absorption mechanism determines how the drug reaches the bloodstream and it is related to the drug physicochemical properties, route of administration and its formulation and design (see Section 1.5. for more details regarding the route of administration and drug formulation). In general, there are different routes for administration of a drug into body, however the most common ones are enteral (e.g. oral), parenteral (e.g. intravenous), inhalation and transdermal.

Each of these ways carries along several advantages and drawbacks. For instance, intravenous infusion (IV) is the most commonly used method amongst the parenteral routes and guarantees an immediate introduction of the drug into the systemic circulation, as the entire dose administered immediately reaches systemic circulation. However, it is not always possible or convenient to use IV administration, for instance in the case of self-administration the application would be difficult. Generally, the oral or the transdermal routes of administrations are preferred by the patients as they present reduced inconvenience.

Following administration through the oral route, the drug enters the gastrointestinal (GI) tract, the most important site of compound absorption. In order to reach the systemic circulation, the drug must cross a significant number of semi-permeable cell membranes. Various types of transport are

identified by which a compound can cross a cell membrane. Firstly, the drug can spread by simple passive diffusion, caused by concentration gradients. Passive diffusion occurs mainly for small molecules with a molecular weight lower than 1000 g/mol. The diffusion rate also depends on other features, such as the molecule's lipid solubility, degree of ionization, and area of absorptive surface of the cell. For instance, small-size molecules tend to penetrate membranes more rapidly than larger ones and as the cell membrane is lipoid, lipid-soluble drugs will also diffuse more rapidly. If the molecule is instead of large dimensions or is ionized, the passage through the cell membrane can take place thanks to facilitated diffusion. In this case the drug is carried with transport proteins, which move due to concentration gradients. Finally, transport proteins can also pass through cell membranes even against concentration gradients, exploiting the chemical energy provided by particular molecules that supply energy to cells, called ATP. This process is called active transport and can lead to higher concentration of the drug in some tissues and organs or preventing transport to others. It is important to note that facilitated diffusion and active transport are selective and saturable processes, i.e. transport for a certain molecule can reach a maximum value depending on the quantity of transporter proteins.

Since melatonin is a lipid soluble molecule with rather small size, it is assumed that it diffuses rapidly through the intestinal walls (Lundquist and Artursson 2016).

In fact, there are several factors that influence the intestinal absorption of drugs including gastric and intestinal pH, drug formulation, solubility, residence times in the lumen, the simultaneous presence of food, and health conditions (Abbiati et al. 2016).

It should be noted that the absorption of the orally administered drug through the stomach and intestine is not guaranteed to be complete, and a fraction of the drug can be expelled unchanged through the faeces. The portion of the drug that reaches the systemic circulation is defined as bioavailability. Bioavailability of a drug is largely determined by the properties of the dosage form which depend partly on its design and manufacture. Individual characteristics such as age, sex, physical activity, genetic phenotype, stress, disorders, or previous GI surgery can also affect drug bioavailability (Porter and Kaplan 2011). In general, different routes of administration lead to different values of bioavailability. As mentioned before, in case of intravenous administration, the drug directly enters the systemic circulation therefore bioavailability equals the whole administered dose. However, if the drug is administered orally, firstly it should pass through the intestinal walls and reach the blood circulatory system.These blood vessels are then collected into the portal vein

metabolism occurs, with a variable extent that depends on the specific drug. The phenomenon where a fraction of the absorbed dose is metabolized before it reaches the systemic blood circulation is aptly called “first-pass effect”. Accordingly, low bioavailability is most common with oral dosage forms especially in case of poorly water-soluble (such as melatonin) and slowly absorbed drugs (Hoffmeister et al. 2012). Another reason for low bioavailability may be an insufficient time for absorption of the drug in the gastrointestinal tract.

Bioavailability is often assessed by determining the area under the plasma concentration-time curve, as illustrated in Figure 11. The area under the curve (AUC) grows with bioavailability as it is proportional to the total amount of unchanged drug that reaches the systemic circulation, thereby accessing the site of action and determining its pharmacological effects.

Figure 11. Representation of the area under the curve (AUC) that is the colored area under the plasma concentration-time profile after oral administration of a drug. AUC is directly proportional to the total

amount of unchanged drug that reaches the systemic circulation (Porter and Kaplan 2011).

When the drug reaches blood, it is distributed to the different organs and tissues through the systemic circulation. In general, the mechanism of distribution is not homogeneous in the organism because it depends on the blood perfusion, tissue binding, local pH, and the permeability of the cell membranes. Moreover, if the diffusion across the membrane is not the limiting step, the distribution equilibrium between blood and tissue is reached faster in the more vascularized areas, i.e. organs and tissues that are highly perfused by blood. Another important aspect that is limiting the drug distribution process is drug binding to plasma protein. Once in the blood, most drugs can bind to plasma proteins (typically human serum albumin, lipoproteins, and globulins). Their mass transfer through capillary exchange is therefore hindered for the protein‐drug ensemble. Thus, only the unbound fraction of drugs is available for passive diffusion out of blood vessels to tissues or organs. This is a distinctive issue that must be carefully considered for dose selection.

The administered drug is cleared by the body either through metabolism, excretion or a combination of both. Drug elimination generally refers to the irreversible removal of a compound or its metabolite(s). The metabolism of compounds generally involves a chemical or enzymatic conversion of the parent compound into one or more metabolites. In particular, the liver is the main site for metabolic reactions in the organism, but further reactions may occur also in the intestine, plasma and tissue cells. In general, drug metabolism converts lipophilic chemical compounds into hydrophilic products which are readily excreted. The excretion of metabolite(s) is mainly facilitated by renal or biliary clearance (Fan and de Lannoy 2014).

Metabolism and excretion occur simultaneously with distribution, hence the ADME processes are dynamic processes and their combined effect provides information about the dynamics of drug concentration in the whole body.

As mentioned earlier, pharmacokinetics attempts to discover the fate of drug from the moment that it is administered up to the point at which is completely eliminated from the body. Thus, pharmacokinetic studies are an essential part of clinical trials and allow the assessment of drugs in terms of safety constraints, feasibility of formulations, delivery strategies and dosage regimens. That is to say, PK studies are important for maximizing therapeutic effects whilst minimizing any drug side effects.

The presence of critical illness alters the disposition of many drugs thus drug concentrations can vary a lot between patients and within the same patient during different phases of the disease. This inter- and intra-individual variability can lead to much more unpredictable pharmacological and toxicological effects. For instance, in patients with organ (e.g. renal, hepatic) dysfunction, drug accumulation may occur, leading to potential side effects. Moreover, administering routine doses defined in non-ICU patients to the ICU population may be problematic, as ICU patients display both PK and PD differences. In order to ensure achieving optimal PK/PD target in ICU patients, it is therefore important to choose dosing strategies that are specific and suitable for this population (Roberts et al. 2016).

In order to determine the most important PK parameters of a drug, practical experiments should be conducted on a number of volunteers/patients by drawing blood samples according to an assigned time schedule. In this manner, it is possible to evaluate the main PK parameters, such as AUC (i.e. area under the drug-concentration- time curve), 𝐶𝑀𝐴𝑋 (i.e. maximum drug concentration), 𝑡𝑀𝐴𝑋 (i.e. time corresponding to 𝐶𝑀𝐴𝑋), 𝑡1/2 (i.e. drug elimination half-life) that is the time required for the