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: \u2022 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). \u2022 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). \u2022 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. \u2022 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. \u2022 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. \u2022 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-1\u3b1 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\ub0C. 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-1\u3b1 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

Transdermal administration of melatonin coupled to cryopass laser treatment as noninvasive therapy for prostate cancer

Melatonin, a pineal gland hormone, exerts oncostatic activity in several types of human cancer, including prostate, the most common neoplasia and the third most frequent cause of male cancer death in the developed world. The growth of androgen-sensitive LNCaP prostate cancer cells in mice is inhibited by 3 mg/kg/week melatonin (0.09 mg/mouse/week) delivered by i.p. injections, which is equivalent to a dose of 210 mg/week in humans. The aim of this study is to test an alternative noninvasive delivery route based on transdermal administration of melatonin onto the tumor area followed by cryo-pass-laser treatment. Two groups of immunodepressed mice were studied, one ( n = 10) subjected to 18 cryopass-laser therapy sessions and one ( n = 10) subjected to the same treatment without melatonin. These groups were compared with mice treated with i.p.-administered melatonin or vehicle with the same time schedule. We found that cryopass-laser treatment is as efficient as i.p. injections in reducing the growth of LNCaP tumor cells, affecting plasma melatonin and redox balance. Furthermore, both delivery routes share the same effects on the involved biochemical pathway driven by hypoxia-inducible factor 1 a . However, cryopass-laser, as used in the present experimental setup, is less efficient than i.p delivery route in increasing the melatonin content and Nrf2 expression in the tumor mass. We conclude that cryopass-laser treatment may have impact for melatonin-based therapy of prostate cancer, by delivering drugs transdermally without causing pain and targeting directly on the site of interest, thereby potentially making long-term treatments more sustainable

Novel strategies to deliver melatonin (in SLN and as cryo-laser therapy) to prostate cancer cells in vivo

Background. Melatonin, synthesized in the pineal gland, modulates malignant cell proliferation via MT1 receptor or dihydrotestosterone-induced calcium influx attenuation. It has also important antioxidant and antiangiogenic properties. Solid-lipid nanoparticles (SLN) is a technology to target drugs against a specific organ, with control of the pharmacokinetics and optimization of their uptake into cancer cells. Cryopass laser therapy consists in the topical application of a frozen drug emulsion followed by a laser scan of the area to give the energy to penetrate the cutaneous barrier and deliver the active principle to the target area. Aim of the study and Methods The aim was to add knowledge on the anticancer action of melatonin with concern to prostate. We used a model of nude mice xenograft of human LNCaP prostate cancer cells and compared the response of the xenografts using up-to-date techniques and different routes to deliver the drug to cancer cells: (a) i.p. as saline; (b) i.p. in SLN; (c) by cryolaser as saline and (d) included in SLN.. Results. Melatonin administered both by i.p. or topically by cryolaser, only 50% of the xenografts resulted in the tumour growth, vs 75% in the control groups. Tumour growth curves showed a similar trend in both groups, but with a marked delay with respect to controls. The mean weight of the tumours collected 45 days after the xenograft was lower in melatonin-treated mice with respect to saline-treated controls (p<0.05). SLN-melatonin did not produce the same inhibition, but histological analysis revealed a different tumour composition. Studies on how the molecular response fits into the observed phenotype are in progress. Conclusions. The results obtained could be the basis for the introduction of this natural molecule as adjuvant active component in therapeutic strategies for the treatment of malignant prostate cancer in humans, and for prevention of cancer relapses

Asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA) and L-arginine in patients with arteriogenic and non-arteriogenic erectile dysfunction

The plasma concentration of asymmetrical dimethylarginine (ADMA), an inhibitor of nitric oxide synthase, has been linked to endothelial dysfunction. We investigated the relation between ADMA, symmetric dimethylarginine (SDMA) and L-arginine concentrations and erectile dysfunction. We compared plasma levels of ADMA, SDMA and L-arginine in 61 men in good health with erectile dysfunction of arteriogenic and non-arteriogenic origin. Diagnosis of erectile dysfunction was based on the International Index of Erectile Function Score and its aetiology was classified with penile echo-colour-Doppler in basal condition and after intracavernous injection of prostaglandin E1. The ADMA and SDMA concentrations were significantly higher in men with arteriogenic erectile dysfunction compared with those with erectile dysfunction of non-arteriogenic origin (p 0.05) nor between each of the two erectile dysfunction subgroups and controls (p > 0.05). The L-arginine/ADMA and the L-arginine/SDMA ratios in arteriogenic erectile dysfunction subgroups were significantly lower than both in controls (p 0.05). We conclude that ADMA and SDMA concentrations are significantly higher and L-arginine/ADMA ratio lower in patients who have arteriogenic erectile dysfunction compared with both patients with non-arteriogenic erectile dysfunction and controls. The negative correlation between ADMA and severity of erectile dysfunction is present only in patients with arteriogenic erectile dysfunction. This study supports the importance to always distinguish arteriogenic from non-arteriogenic erectile dysfunction patients to study the complicate erectogenic mechanisms that lead to erectile dysfunction and also to provide potential therapeutic agents for patients with arteriogenic erectile dysfunction

Melatonin : positive effects on high-risk patients with desynchronized sleep-wake rhythm

Background Melatonin (MT), a pineal gland hormone, mediates many processes, including the biorhythmic regulation of the organism physiology. The role of MT in the treatment of sleep disturbances, to prevent jet lag or as a part of the sepsis treatment is widely discussed. Circadian rhythm of MT is desynchronized in critically ill pts in intensive care unit (ICU). The restoration of MT levels has been recently proved to be useful. The aims of the investigation are: to confirm that MT is a molecule active in the regulation of sleep/wake rhythm in ICU patients and to compare the differences in the MT pharmacokinetics using different administration route and drug formulation. Methodology The clinical effects of long term (5 days) administration of oral MT, as well as the pharmacokinetics profiles as a function of different administration ways (os, os by SLN (solid lipid nanoparticles), transdermal by SLN) have been studied in ICU pts. From the second day of the ICU stay, serial withdrawal were taken to determine both the endogenous and the exogenous plasma MT content, for a total of 20 withdrawal for each patient. Each blood sample was centrifuged and the plasma stored at -20\ub0C. To determine the MT concentration we used an ELISA kit that includes a pre-purification of the sample by SPE (solid phase extraction) cartridges. Results In this study, we have seen that administration of oral MT, is safe, reduces need for analgesic and sedative drugs and shows better neurological status indicators, also restoring the normal circadian rhythm. In patients who have received oral MT, the absorption is rapid: the peak plasma concentration has a median of 30 minutes and after only 5 minutes the MT levels were significantly higher than physiological ones. The group treated with transdermal MT presents a delayed peak plasma concentration (4 h). Conclusions In this study, we have proved that MT 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 MT levels as well as enteral administration and is more practicable in clinical setting

Novel strategies to deliver melatonin (in SLN and by cryo-laser therapy) to prostate cancer cells

Background: Melatonin (MT) is a chemical signal of dark/light, and serves as a bioclock and bio-calendar to mediate many receptor- or non receptor-mediated functions. MT also has a relevant oncostatic activity, especially with respect to prostate cancers, recently related to hypoxia and sphingolipids signaling pathways. The aims of the investigation are: to confirm that MT is active in the cure of prostate cancer, to speculate on the underlying mechanisms, to investigate the signaling pathways involved and to assess whether alternative and novel ways to deliver the drug may be competitive. Methods: We used an in vivo model of human prostate tumor LNCaP cells xenografted into nude athymic mice. MT has been administered i.p. as saline (n=13) and by SLN (solid lipid nanoparticles) (n=13) or transdermally by Cryopass therapy (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. MT concentration was assessed in plasma and tissues by ELISA test and tumors were evaluated for morphology, MT content and HIF-1a expression. Results: Tumors developed slowly in all the MT-treated (topical and i.p.) groups and at the end of the treatment, the mean volume was significantly lower vs control. Both tumor and plasma levels were significantly higher in treated vs not-treated animals. Harvested tumor showed a strong inflammatory reaction which seemed to surround and infiltrate the tumor cells. In SLN-MT 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-1 expression increased in both treatment groups vs control. Conclusions: We have confirmed the positive effects of MT 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 MT are effective as well. This would accelerate the transferability of obtained data towards a therapy. on MT oncostatic activity