MELATONIN: A PLEIOTROPIC MOLECULE OF NATURAL ORIGIN. EVALUATION OF THE DIFFERENT THERAPEUTIC ACTIVITIES IN ANIMAL MODELS AND / OR HUMAN PATIENTS AND A STUDY OF THE METABOLIC-BIOCHEMICAL PATHWAYS RELATED TO THEM.

1.0. ABSTRACT Background Melatonin (MLT), a pineal gland hormone, seves as a bioclock and bio-calendar to mediate many receptor- or non-receptor functions. In addition to its immunomodulatory and neurological effects, MLT has a relevant oncostatic activity especially with respect to breast and prostate cancers, but the mechanism of action is still unclear. The growth of androgen-independent LNCaP prostate cancer cells has been demonstrated to be inhibited by MLT both in vitro and in vivo in a nude mice xenograft model. Clearly, the oncostatic effects of MLT may not be related to a single function, but rather to a complex interaction of several factors that involve the redox state, the immune system, the modulation of the endocrine system and membrane receptors. MLT also increases sleepiness, decreases core temperature and increases peripheral temperature in humans. The role of MLT in the treatment of sleep disturbances, to prevent jet lag or as a part of the sepsis treatment is widely discussed; yet the role in critically ill patients still deserves further investigation. Critically ill patients suffer from severe sleep disturbances during their stay in an Intensive Care Unit (ICU). Moreover, these patients require high levels of antioxidants due to their critical illness. Aims of the thesis The main object of my PhD thesis was to confirm the pleiotropy of MLT molecule by testing its activity in two of the most promising clinical applications: the cure of prostate cancer and the regulation of the sleep/wake rhythm as adjuvant in the sedative therapy in critically ill patients. Spcific Aims: • To evaluate the oncostatic effect of MLT administered intraperitoneally (i.p.) by saline solution on human prostate tumor. To this purpose I have selected an in-vivo experimental model of nude mice (athymic), xenografted subcutaneously with tumor cells of a human prostatic line (LNCaP). • Using the same animal model and the same administration route (i.p.) and treatment schedule of MLT administered in saline, to investigate the efficacy of a novel and promising pharmaceutical formulation: MLT included in a solid lipid nanoparticles system (SLN-MLT). • Using the same mouse model of human prostate cancer, to test whether MLT can be administered efficiently using alternative ways that are more sustainable for prolonged treatments than i.p. MLT, e.g., transdermal delivery through the skin barrier directly onto the tumor via a novel and patented technique named cryoRx. • To focus on the underlying action mechanism of MLT at the tumor cellular micro-environment and the possible influence on such a mechanism of the lipid nanocarrier employed. • To evaluate in a cohort of ICU patients, if the circadian rhythm of MLT secretion is disrupted and to which extent MLT administration by different routes and different drug formulations (MLT as a tablet administered os, MLT encapsulated in SLN administered os as a suspension and MLT encapsulated in SLN applied transdermally as a suspension with the aid of a patch) is feasible in terms of absorption efficiency and adequacy in achieving and maintaining nocturnal peak plasma hormone. • To evaluate if the restoration of the melatoninemia by the different ways of drug delivery in critically ill patients may be useful to restore the pleiotropic function of this hormone: facilitate the resolution of sleep-wake cycle disorders, improve the quality of sleep, reduce the number of episodes of anxiety, confusion and agitation, and reduce the amount of sedatives used, especially at night. Materials and Methods We used an in vivo model of human prostate tumor LNCaP cells xenografted into nude athymic mice. MLT has been administered i.p. as saline (n=13) and by SLN (n=13) or transdermally by cryoRx (n=14). For each treatment controls were also included. Each group received the same administration schedule: 3 treatments per week, for 6 week. At the end the animals were sacrificed and along the treatment period the mice weight were recorded as well as the tumor volume was measured. MLT concentration was assessed in plasma and tissues by ELISA test and tumors were evaluated for morphology, MLT content and HIF-1α expression. The clinical effects of MLT administration as well as the pharmacokinetics profiles as a function of different administration ways (oral as MLT, oral as SLN and transdermal as SLN) have been studied in ICU patients. During the 2nd day of the ICU stay, serial withdrawal were taken to determine the endogenous MLT secretion, and then after MLT administration, additional plasma samples were obtained during the 3rd day to evaluate the exogenous plasma MLT content, for a total of 20 withdrawal for each patient. Each blood sample was centrifuged and the plasma stored at -20°C. To determine the MLT concentration we used an ELISA kit that includes a pre-purification of the sample by SPE (solid phase extraction) cartridges. Results Tumors developed slowly in all the MLT-treated (topical and i.p.) groups and at the end of the treatment, the mean volume was significantly lower vs control. Both tumoral and plasma MLT levels were significantly higher in treated (topical and i.p.) vs not-treated animals. Harvested tumor showed a strong inflammatory reaction which seemed to surround and infiltrate the tumor cells. In SLN-MLT treated animals, in addition to a strong lymphocyte infiltration, the tumor appeared limited also by the presence of fibroblast type cells. Preliminary results showed HIF-1α expression increased in both treatment groups (topical and i.p.) vs Ctrl. In the clinical study, we have seen that MLT administration, is safe, reduces need for analgesic and sedative drugs restoring the normal circadian rhythm. In patients who received MLT or SLN-MLT by os, the absorption was rapid: the peak plasma concentration had a median of 30 min and after only 5 min, the MLT levels were significantly higher than physiological ones. The AUC of SLN-MLT was significantly higher than when MLT was administered by saline solution. SLN-MLT by transdermal route, presented a delayed peak plasma concentration (4 h) and a lower bioavailability but MLT plasma levels reached however the pharmacological concentration able to restore the pleiotropic function of this hormone and facilitate the resolution of sleep-wake cycle disorders. Conclusions We have confirmed the positive effects of MLT on tumor growth and we have focused on its effect on hypoxia. The possible role as anti-tumor drug candidate deserves to be further investigated. We demonstrated that different alternative and novel ways to deliver MLT are effective as well. This would accelerate the transferability of obtained data towards a therapy. on MLT oncostatic activity. In the clinical study, we have proved that MLT is able to normalize the sleep-wake cycle, to ameliorate the sleep quality and to reduce the number of sedative drugs used in ICU pts. We proved also that transdermal administration by SLN is effective in rising plasma MLT levels as well as enteral administration and is more practicable in clinical setting.