Daniele Esposti

PHARMACOLOGY OF SILYMARIN

The flavonoid silymarin and one of its structural components, silibinin, are substances with documented hepatoprotective properties. Their mechanisms of action are still poorly understood. However, the data in the literature indicate that silymarin and silibinin act in four different ways: (i) as antioxidants, scavengers and regulators of the intracellular content of glutathione; (ii) as cell membrane stabilisers and permeability regulators that prevent hepatotoxic agents from entering hepatocytes; (iii) as promoters of ribosomal RNA synthesis, stimulating liver regeneration; and (iv) as inhibitors of the transformation of stellate hepatocytes into myofibroblasts, the process responsible for the deposition of collagen fibres leading to cirrhosis. The key mechanism that ensures hepatoprotection appears to be free radical scavenging. Anti-inflammatory and anticarcinogenic properties have also been documented.Silymarin is able to neutralise the hepatotoxicity of several agents, including Amanita phalloides, ethanol, paracetamol (acetaminophen) and carbon tetrachloride in animal models. The protection against A. phalloides is inversely proportional to the time that has elapsed since administration of the toxin. Silymarin protects against its toxic principle α-amanitin by preventing its uptake through hepatocyte membranes and inhibiting the effects of tumour necrosis factor-α, which exacerbates lipid peroxidation.Clinical trials have shown that silymarin exerts hepatoprotective effects in acute viral hepatitis, poisoning by A. phalloides, toxic hepatitis produced by psychotropic agents and alcohol-related liver disease, including cirrhosis, at daily doses ranging from 280 to 800mg, equivalent to 400 to 1140mg of standardised extract. Hepatoprotection has been documented by improvement in liver function tests; moreover, treatment with silymarin was associated with an increase in survival in a placebo-controlled clinical trial in alcoholic liver disease.Pharmacokinetic studies have shown that silymarin is absorbed by the oral route and that it distributes into the alimentary tract (liver, stomach, intestine, pancreas). It is mainly excreted as metabolites in the bile, and is subject to enterohepatic circulation. Toxicity is very low, the oral 50% lethal dose being 10 000 mg/kg in rats and the maximum tolerated dose being 300 mg/kg in dogs. Moreover, silymarin is devoid of embryotoxic potential.In conclusion, silymarin is a well tolerated and effective antidote for use in hepatotoxicity produced by a number of toxins, including A. phalloides, ethanol and psychotropic drugs. Numerous experimental studies suggest that it acts as a free radical scavenger, with other liver-specific properties that make it a unique hepatoprotective agent.

A clinical study on the relationship between the pineal gland and the opioid system

Recent reports point to a link between the pineal gland and the opioid system. In order to investigate this relationship, two separate studies were performed on humans. Beta-endorphin plasma levels were determined after melatonin administration (0.2 mg/kg b.w. i.m. at 2 p.m.). Melatonin serum values were evaluated after administration of FK 33-824, a met-enkephalin analogue (0.3 mg i.v. infusion at 9 a.m.). A significant decrease of beta-endorphin plasma levels was observed 120 minutes after melatonin injection. Melatonin release was stimulated by FK 33-824, with a peak at 30 minutes. The present results provide evidence of the inhibitory effect of melatonin on beta-endorphin secretion and the stimulatory action of the opioid peptides on the pineal gland. However, further studies will be required to clarify the relationship between the opioid system and the pineal gland.

The natural antioxidant alpha-lipoic acid induces p27Kip1-dependent cell cycle arrest and apoptosis in MCF-7 human breast cancer cells

Unlike normal cells, tumor cells survive in a specific redox environment where the elevated reactive oxygen species contribute to enhance cell proliferation and to suppress apoptosis. Alpha-lipoic acid, a naturally occurring reactive oxygen species scavenger, has been shown to possess anticancer activity, due to its ability to suppress proliferation and to induce apoptosis in different cancer cell lines. Since at the moment little information is available regarding the potential effects of alpha-lipoic acid on breast cancer, in the present study we addressed the question whether alpha-lipoic acid induces cell cycle arrest and apoptosis in the human breast cancer cell line MCF-7. Moreover, we investigated some molecular mechanisms which mediate alpha-lipoic acid actions, focusing on the role of the PI3-K/Akt signalling pathway. We observed that alpha-lipoic acid is able to scavenge reactive oxygen species in MCF-7 cells and that the reduction of reactive oxygen species is followed by cell growth arrest in the G1 phase of the cell cycle, via the specific inhibition of Akt pathway and the up-regulation of the cyclin-dependent kinase inhibitor p27(kip1), and by apoptosis, via changes of the ratio of the apoptotic-related protein Bax/Bcl-2. Thus, the anti-tumor activity of alpha-lipoic acid observed in MCF-7 cells further stresses the role of redox state in regulating cancer initiation and progression.

Action of morphine on melatonin release in the rat

Some data from the literature raised the possibility of an interaction between the opioidergic system and pineal secretion. The present study was undertaken in order to investigate the acute influence exerted by opioids upon plasma melatonin levels in the albino rat. Different doses of morphine hydrocloride were injected (1, 1.5, 2, 3 mg/kg) intraperitoneally into anaesthetized adult male rats bearing a cannula previously inserted into the carotid. Blood samples were collected subsequently at 30‐min intervals, within a period of 90 min following drug administration. Plasma melatonin contents were determined by a radioimmu‐noassay (RIA) method. Acute administration resulted in a dose‐dependent increase in plasma melatonin concentration when compared to the respective controls. This effect is blocked by pretreatment with Naloxone. The present result seem to support the hypothesis that the opioidergic system, in certain circumstances, might contribute to the activation of melatonin secretion.

Melatonin involvement in immunity and cancer

The most studied endocrine product of the pineal gland, melatonin, has been reported to be involved in the feedback between neuroendocrine and immune functions and to exert oncostatic action, at least in certain experimental conditions. Melatonin seems to be an integral part of the immune system, by exerting direct and/or indirect stimulatory effects on both cellular and humoral immunity. Likewise, an antitumor activity of melatonin has been shown in several experimental models in vivo and in vitro. The means by which melatonin exerts its effects on immunity and neoplastic growth have not been elucidated. The different putative mechanisms of action of melatonin investigated so far are here briefly discussed.

Clinical Results with the Pineal Hormone Melatonin in Advanced Cancer Resistant to Standard Antitumor Therapies

The pineal hormone melatonin (MLT) is able to exert an oncostatic action. Its possible use in the treatment of human tumors, however, has not yet been investigated. The present study was carried out to evaluate the effects of MLT in patients with metastatic solid tumors resistant to conventional therapies. The study included 54 patients, most of them were affected by lung cancer or colorectal carcinoma. MLT was given intramuscularly at a daily dose of 20 mg at 3.00 p.m. for 2 months; this induction phase was followed by a maintenance period at a dose of 10 mg orally in responder patients or in those with an improvement in performance status (PS). The clinical response was as follows: 1 partial response (cancer of pancreas), 2 minor responses (colon cancer and hepatocarcinoma) and 21 with stable disease. The remaining 30 patients rapidly progressed within the first 2 months of therapy. An evident improvement in PS was achieved in 18 of 54 (33%) cases. These results, by showing an apparent control of the neoplastic growth and an improvement in the quality of life in a reasonable number of cancer patients for whom no other standard therapy is available, would justify further clinical trials to better define the impact of MLT therapy on the survival and quality of life of untreatable advanced cancer patients.

A clinical study of the pineal gland activity in oncologic patients

It has been known for many years that the pineal gland is involved in regulating tumor growth. In order to evaluate the functional activity of the pineal gland in neoplastic diseases, melatonin serum levels and its light/dark rhythm have been determined with the RIA method in patients affected by various forms of tumor. Irrespectively of the type of the tumor and of its localization, existence of two subpopulations has been observed within the oncologic patients, the former with normal levels of melatonin, and the latter with high ones. The light/dark rhythm of melatonin was anomalous in some cases. An evident decrease of serum melatonin values was seen after chemotherapy. It might be interesting to establish whether melatonin levels may conditionate the prognosis of patients with cancer.

Melatonin Influences Human Balance

In order to evaluate a possible correlation between melatonin, the cerebellum and, consequently, human balance, a double-blind pilot study was performed in 5 subjects with random administration of different doses of melatonin. Before and 1 h after a single administration, a complete otoneurological examination was performed. This first pilot study revealed that melatonin had effects on human equilibrium although these effects were not dosage related and were different in individual subjects. On the basis of these results, a second study was performed. Fourteen healthy volunteers were investigated before and 1 h after administration of a single dose of 10 mg melatonin. The otoneurological examination was restricted to the evaluation of: horizontal saccades, horizontal sinusoidal smooth pursuit, eyes open, eyes closed and head retroflexed static posturography. All subjects showed a decrease in posturographic performances, especially in the simplest test (eyes open) and half of them (6 out of 13) showed also impairment of eye movements. These results confirm the role of melatonin in the control of sensorimotor performances, and the cerebellar receptors might be correlated with the control of human balance.

Clinical study of melatonin in untreatable advanced cancer patients.

It is known that the pineal gland has some antitumor activity. Melatonin, its most important hormone, has been shown to inhibit tumor growth in vivo and in vitro. Moreover, some investigations have demonstrated an altered melatonin secretion in cancer patients. Despite these interesting data, clinical trials have never been carried out to evaluate the effects of melatonin on human neoplasms. The aim of this study was to draw some preliminary conclusions on melatonin therapy in advanced human neoplasms. Nineteen patients suffering from advanced solid tumors, which did not respond to standard therapies, entered the study. Performance status (PS) was 20 or less in 9 cases, and more than 20 in the other 10. Melatonin was given intramuscularly at a daily dose of 20 mg at 3.00 p.m., followed by a maintenance period with lower doses in patients who had a remission, a stabilization of disease or an improvement in PS. Among patients with a PS higher than 20, a partial response was achieved in one case with cancer of the pancreas; moreover, 5 of 10 had stable disease, but the other 4 cases had a progression; an evident improvement of PS was obtained in 6 of the 10 cases. In contrast, among patients with a very poor PS, 7 of 9 died within the first 2 months of therapy. This preliminary study would suggest that melatonin may be of some value in treating cancer patients in whom standard antitumor therapies have failed, particularly in improving their PS and quality of life.

A new melatonin receptor ligand with mt1‐agonist and MT2‐antagonist properties

It has been difficult, so far, to obtain melatonin analogs possessing high selectivity for the respective melatonin receptors, mt1 and MT2. In the present work, we report the synthesis and pharmacological characterization of a new compound N‐{2‐[5‐(2‐hydroxyethoxy)‐1H‐indol‐3‐yl)] ethyl} acetamide or 5‐hydroxyethoxy‐N‐acetyltryptamine (5‐HEAT). To assess the activity of the compound, the following tests were performed: affinity determination for the high‐ and low‐affinity receptor states (2‐[ I]iodomelatonin binding), potency and intrinsic activity in inducing G protein activation ([ S]GTPγS binding assay). 5‐HEAT showed little selectivity for the mt1 receptor, with pKi values of 7.77 for mt1 and 7.12 for the MT2 receptors, respectively. 5‐HEAT was able to differentiate between the high‐ and the low‐affinity receptor states in the mt1 but not in the MT2 receptor. 5‐HEAT induced a high level of G protein activation when acting through the mt1 receptor, with a relative intrinsic activity of 0.92. On the contrary, it elicited only minimal MT2 receptor‐mediated G protein activation, with a relative intrinsic activity of 0.16, and was also able to inhibit the melatonin‐induced MT2 receptor‐mediated G protein activation, with a pKB value of 7.4. In conclusion, it appears that 5‐HEAT possesses very different efficacies at the two melatonin receptors, behaving as a full melatonin receptor agonist at the mt1 and as an antagonist/weak partial agonist at the MT2 receptor. Therefore, it is a promising ligand for use in functional studies aimed at distinguishing between the effects mediated by the different melatonin receptors in the human.