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‘Burnout syndrome’ is a progressive mental process whereby individuals are unable to adapt to prolonged and excessive stress at work. The main features of the syndrome are physical and emotional fatigue, depersonalization and frustration with ineffective professional and personal implementation. The first studies carried out in the 1970’s showed that this condition is much more common in the social professions employing teachers, doctors, nurses, social workers and police officers. Currently, the Maslach Burnout Inventory (MBI) is the most important medical research tool for diagnosing level of burnout. Treatment of burnout depend on the severity of the syndrome. In severe forms, in addition to socio-occupational cognitive behavioural therapy, the approach also includes drug treatment with benzodiazepines and antidepressants, particularly the selective serotonin reuptake inhibitors.

Panax ginseng is traditionally used for enhancement of physical capacities, especially in condition of severe fatigue. To evaluate the ability of ginseng in enhancing performance, we focused on the gene expression of AMPK, PGC-1α and SIRT1 genes, involved in energy balance. Gene expression was evaluated in C2C12 myotubes, using P. ginseng C.A. Meyer standardized extract G115, at various concentrations (10-50-100-200 mg/L) for 24, 48, 72 h of incubation. G115 increased significantly expression of AMPK and PGC-1α in dose and time-dependent manner: at the concentration of 10 mg/L there is an increase after 72 h of incubation while at 50 mg/L after 48 h and maintained for 72 h. G115 increased expression of SIRT1 at 10 mg/L after 24 h, while at 50 mg/L after 24 and 48 h. The treatment enhanced cellular ATP levels and O2 consumption only at 50 mg/L. We investigated AMPK, PGC-1α and SIRT1 expression in gastrocnemius muscle during acute and prolonged exercise in mice treated with G115. G115 showed no effect on acute exercise compared to untreated mice, but after prolonged exercise there were significant increases of AMPK and PGC-1α. Expression of SIRT1 was also significant in treated animals’ undergone prolonged exercise. Our data indicate that treatment with G115 increases AMPK and SIRT1 expression that may be related to the induction of PGC-1α expression which improves the energy balance, as demonstrated by increased production of ATP and oxygen consumption. In addition, G115 is able to counteract the inhibition of gene expression of AMPK and PGC-1α due to the prolonged exercise.

New derivatives of 4-phenyl-2-propionamidotetralin (4-P-PDOT) were prepared and tested on cloned MT1 and MT2 receptors, with the purpose of merging previously reported pharmacophores for nonselective agonists and for MT2-selective antagonists. A 8-methoxy group increases binding affinity of both (±)-cis- and (±)-trans-4-P-PDOT, and it can be bioisosterically replaced by a bromine. Conformational analysis of 8-methoxy-4-P-PDOT by molecular dynamics, supported by NMR data, revealed an energetically favored conformation for the (2S,4S)-cis isomer and a less favorable conformation for the (2R,4S)-trans one, fulfilling the requirements of a pharmacophore model for nonselective melatonin receptor agonists. A new superposition model, including features characteristic of MT2-selective antagonists, suggests that MT1/MT2 agonists and MT2 antagonists can share the same arrangement for their pharmacophoric elements. The model correctly predicted the eutomers of (±)-cis- and (±)-trans-4-P-PDOT. The model was validated by preparing three dihydronaphthalene derivatives, either able or not able to reproduce the putative active conformation of 4-P-PDOT.

We report the synthesis, binding properties and intrinsic activity at MT(1) and MT(2) melatonin receptors of new dimeric melatonin receptor ligands in which two units of the monomeric agonist N-{2-[(3-methoxyphenyl)methylamino]ethyl}acetamide (1) are linked together through different anchor points. Dimerization of compound 1 through the methoxy substituent leads to a substantial improvement in selectivity for the MT(1) receptor, and to a partial agonist behavior. Compound 3a, with a trimethylene linker, was the most selective for the MT(1) subtype (112-fold selectivity) and compound 3d, characterized by a hexamethylene spacer, had the highest MT(1) binding affinity (pK(iMT1)=8.47) and 54-fold MT(1)-selectivity. Dimerization through the aniline nitrogen of 1 abolished MT(1) selectivity, leading to compounds with either a full agonist or an antagonist behavior depending on the nature of the linker.

Nosocomial pneumonia (NP) is associated with considerable morbidity and mortality. Data have shown that inadequate initial antibiotic therapy is a major risk for infection-attributed mortality. The aim of the present study was to measure antibiotic concentration and minimum inhibitory concentration (MIC) in infected hospitalised patients early in therapy, in order to determine whether dose alterations, in those with low drug concentrations, could affect outcomes. Only patients treated with aminoglycosides, fluoroquinolones, and beta-lactams were evaluated. MICs were determined using standard National Committee for Clinical Laboratory Standards procedures. Antibiotics were assayed using validated high-performance liquid chromatographic methods. Pharmacokinetic/pharmacodynamic markers adopted were: aminoglycoside peak/MIC ratio >or=8 mg L(-1); fluoroquinolone peak/MIC >or=10 mg L(-1); beta-lactam peak/MIC >or=4 mg L(-1) and time that plasma levels remain above the MIC >or=70%. 638 patients with NP were included in the study. In 205 patients, both drug concentration and isolate MIC were available, while in other patients, used as controls, one or both parameters were lacking. For clinical outcome, the Acute Physiology and Chronic Health Evaluation II score (p

Background:Serenoa repens extract is the phytotherapeutic agent most frequently used for the treatment of the urological symptoms caused by benign prostatic hyperplasia. There are many extracts in the market and each manufacturer uses different extraction processes; for this reason, it’s possible that one product is not equivalent to another. The aim of this study was to compare the activity of different extracts of Serenoa repens marketed in Italy. Methods: The following extracts were tested on 10 day co-cultured epithelial and fibroblast cells by a 5α-reductase activity assay: Permixon®, Saba®, Serpens®, Idiprost®, Prostamev®, Profluss® and Prostil®. In order to assess the variability in Serenoa repens products, 2 different batches for each brand were evaluated. Results and Conclusions: All extracts tested, albeit variably, are able to inhibit both isoforms of 5α-reductase. However, the potency of the extracts appears to be very different, as well as the potencies of 2 different batches of the same extract. This is probably due to qualitative and quantitative differences in the active ingredients. So, the product of each company must be tested to evaluate the clinical efficacy and bioactivity.

The effect of Panax ginseng C.A. Meyer G115 on inflammatory cytokine production and toll-like receptor 4 (TLR4) RNA expression was examined in mice during 4 weeks of swimming stress. Mice were assigned to four groups: (1) control (no exercise); (2) control-G115 (25 mg/kg/day p.o.); (3) stress (kept swimming for 60 min daily); and (4) stress-G115 (25 mg/kg/day p.o. and kept swimming for 60 min daily). Peritoneal macrophages were collected at rest each week. RNA was extracted and processed for real-time PCR. An aliquot of macrophages was lipopolysaccharide (LPS)-stimulated for TNF-alpha and IL-1beta production. Different expression patterns between untreated and treated groups, and between TLR2 and TLR4 were found. High levels of TLR4 expression in the control-G115 group were detectable significantly at the first, and at the second week (P

Herein we report attempts to optimize the pharmacological properties of 5-(2-hydroxyethoxy)-N-acetyltryptamine (5-HEAT), a melatonin receptor ligand previously described by us. Several 5-substituted and 2,5-disubstituted N-acyltryptamines were synthesized and evaluated in vitro for the human cloned MT(1) and MT(2) receptors. From this series of N-acyltryptamines the 2-bromo derivative (5 c) retains the interesting efficacy profile of 5-HEAT and shows increased melatonin receptor affinities; it represents one of the first examples of a high-affinity MT(1) agonist/MT(2) antagonist. Some other full agonists for both melatonin receptors which exhibit similar or increased affinity relative to that of melatonin were obtained.

Chlamydophila pneumoniae is a pathogen that is involved in acute and chronic respiratory infections and that is associated with asthma and coronary artery diseases. In this study, we evaluated the effects of PEX, a noncatalytic metalloproteinase fragment with integrin-binding activity, against experimental infections caused by C. pneumoniae . Moreover, we investigated the relationships between C. pneumoniae and α v β 3 integrin functions in order to explain the possible mechanism of action of PEX both in vitro and in vivo. For the in vitro experiments, HeLa cells were infected with C. pneumoniae and treated with either PEX or azithromycin. The results obtained with PEX were not significantly different ( P > 0.05) from those achieved with azithromycin. Similar results were also obtained in a lung infection model. Male C57BL/J6 mice inoculated intranasally with 10 6 inclusion-forming units of C. pneumoniae were treated with either PEX or azithromycin plus rifampin. Infected mice treated with PEX showed a marked decrease in C. pneumoniae counts versus those for the controls; this finding did not differ significantly ( P > 0.05) from the results observed for the antibiotic-treated group. Integrin α v β 3 plays an important role in C. pneumoniae infection. Blockage of integrin activation led to a significant inhibition of C. pneumoniae infection in HeLa cells. Moreover, CHO DHFR α v β 3 -expressing cells were significantly ( P < 0.001) more susceptible to C. pneumoniae infection than CHO DHFR cells. These results offer new perspectives on the treatment of C. pneumoniae infection and indicate that α v β 3 could be a promising target for new agents developed for activity against this pathogen.

3-(Acetylaminomethyl)-2-(ethoxycarbonyl)-6-methoxy-1,3,4,5-tetrahydrobenzo[cd]indole (2) is a rigid melatonin analogue that as a racemate displays about the same affinity and intrinsic activity of melatonin (1) in in vitro experiments. We report here the resolution of the racemate by preparative medium pressure liquid chromatography (MPLC) and the X-ray determination of the R absolute configuration of the (-)-enantiomer. The two enantiomers were separately tested as MT1 and MT2 ligands, and the (+)-(S)-2 showed a potency comparable to that of melatonin and about three orders of magnitude greater than that of its enantiomer. The information obtained by crystallographic analysis and NMR studies about the conformational preference for 2 and by the pharmacological characterization of (R)-2 and (S)-2 was employed in a molecular modeling study, aimed at reassessing the melatonin receptor pharmacophore model for agonist compounds. Chiral enantioselective agonists reported in the literature were also included in the study.

Ginseng (Panax ginseng C.A. Meyer, ginseng) has been the most precious and renowned tonic drug in traditional Chinese medicine. Although the original discovery of its therapeutic efficacy has been lost in antiquity, believers in ginseng as a panacea have sought, cultivated, preserved and extracted its essences for the treatment of a wide range of ailments. Many patients have used aqueous and alcoholic extracts of ginseng during convalescence, particularly where previous health problem(s) resulted in some degree of debilitation, and a corresponding deficit in mental, physical or other functional capacities. An impressive body of information has been accumulated and scientific research has documented and reviewed the useful effects of P. ginseng C.A. Meyer. The high variability in composition of the marketed products clearly affects the results of clinical studies. The availability of the standardised ginseng extract G115® has made it possible to generate reproducible results in animal studies and human clinical research.

Several indole analogues of melatonin (MLT) were obtained by moving the MLT side chain from C(3) to C(2) of the indole ring. Binding and in vitro functional assays were performed on cloned human MT1 and MT2 receptors, stably transfected in NIH3T3 cells. Quantitative structure-activity relationship studies showed that 4-methoxy-2-(N-acylaminomethyl)indoles, with a benzyl group in position 1, were selective MT2 antagonists and, in particular, N-[(1-p-chlorobenzyl-4-methoxy-1H-indol-2-yl)methyl]propanamide (12) behaved as a pure antagonist at MT1 and MT2 receptors, with a 148-fold selectivity for MT2. We present a topographical model that suggests a lipophilic group, located out of the plane of the indole ring of MLT, as the key feature of the MT2 selective antagonists.

A number of 6-methoxy-1-(2-propionylaminoethyl)indoles, carrying properly selected substituents at the C-2 indole position, were prepared and tested as melatonin receptor ligands. Affinities and intrinsic activities for the human cloned mt 1 and MT 2 receptors were examined and compared with those of some 2-substituted melatonin derivatives recently described by us. A quantitative structure–activity relationship (QSAR) study of the sixteen 2-substituted indole compounds, 5a – k , 1 , 8 – 11 , using partial least squares (PLS) and multiple regression analysis (MRA) revealed the existence of an optimal range of lipophilicity for the C-2 indole substituent. There are also indications that planar, electron-withdrawing substituents contribute to the affinity by establishing additional interactions with the binding pocket. No mt 1 /MT 2 subtype selectivity was observed, with the relevant exception of the 2-phenethyl derivative 5e , which exhibited the highest selectivity for the h-MT 2 receptor among all the compounds tested (MT 2 /mt 1 ratio of ca. 50). Conformational analysis and superposition of 5e to other reported selective MT 2 ligands revealed structural and conformational similarities that might account for the MT 2 /mt 1 selectivity of 5e .

It has been difficult, so far, to obtain melatonin analogs possessing high selectivity for the respective melatonin receptors, mt 1 and MT 2 . 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-[ 125 I]iodomelatonin binding), potency and intrinsic activity in inducing G protein activation ([ 35 S]GTPγS binding assay). 5-HEAT showed little selectivity for the mt 1 receptor, with pK i values of 7.77 for mt 1 and 7.12 for the MT 2 receptors, respectively. 5-HEAT was able to differentiate between the high- and the low-affinity receptor states in the mt 1 but not in the MT 2 receptor. 5-HEAT induced a high level of G protein activation when acting through the mt 1 receptor, with a relative intrinsic activity of 0.92. On the contrary, it elicited only minimal MT 2 receptor-mediated G protein activation, with a relative intrinsic activity of 0.16, and was also able to inhibit the melatonin-induced MT 2 receptor-mediated G protein activation, with a pK B 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 mt 1 and as an antagonist/weak partial agonist at the MT 2 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.

NIH3T3 fibroblast cells transfected with the full-length coding region of the MT2 human melatonin receptor stably expressed the receptor that is coupled to a pertussis toxin-sensitive G protein and exhibits high affinity for melatonin (KI=261 pM). The order of apparent affinity for selected compounds was: 4-phenyl-2-propionamidotetralin (4P-PDOT)>2-phenylmelatonin>2-iodomelatonin>2-bromomelatonin>6-chloromelatonin⩾melatonin>luzindole>N-acetyl-tryptamine⩾N-[(2-phenyl-1H-indol-3-yl)ethyl]cyclobutanecarboxamide (compound 6)>N-acetylserotonin. 4P-PDOT exhibited a very high selectivity (∼22,000 times) for the MT2 receptor with respect to the mt1 receptor subtype, as tested in comparative experiments with membrane preparations from NIH3T3 cells stably transfected with the human mt1 receptor. MT2 melatonin receptors mediated incorporation of [35S]-GTPγS into isolated membranes via receptor catalyzed exchange of [35S]-GTPγS for GDP. The relative intrinsic activity and potency of the compounds were subsequently studied by using [35S]-GTPγS incorporation. The order of potency was equal to the order of apparent affinity. Melatonin and full agonists increased [35S]-GTPγS binding by 250% over basal (taken as 100%). Luzindole did not increase basal [35S]-GTPγS binding but competitively inhibited melatonin-stimulated [35S]-GTPγS binding, thus exhibiting antagonist action. The other two mt1 antagonists used here, 4P-PDOT and N-[(2-phenyl-1H-indol-3-yl)ethyl]cyclobutanecarboxamide, behaved as partial agonists at the MT2 subtype, with relative intrinsic activities of 0.37 and 0.39, respectively. These findings show, for the first time, important differences in the intrinsic activity of analogues between the human mt1 and MT2 melatonin receptor subtypes. Keywords: Melatonin, MT2 receptor, melatonin, relative efficacy, melatonin analogues, melatonin antagonists, [35S]-GTPγS binding Introduction Melatonin is a hormone secreted rhythmically by the mammalian pineal gland. This hormone plays a central role in the regulation of many types of circadian and seasonal behaviour in mammals (Morgan et al., 1994). In man, melatonin elicits circadian, cardiovascular and hypnotic effects (Reppert et al., 1996; Dollins et al., 1994; Lewy et al., 1995; Cagnacci et al., 1998). Melatonin appears to evoke its effects through high affinity, G protein-coupled receptors (Morgan et al., 1994). Currently, two subtypes of human melatonin receptors have been identified: mt1 (preferentially expressed in the brain) and MT2 (preferentially expressed in the retina) (Reppert et al., 1994; 1995). Both native and recombinant melatonin receptors are negatively coupled to adenylyl cylase by pertussis toxin-sensitive G proteins (Morgan et al., 1994; Reppert et al., 1994; 1995). Little is known about the relative roles of the two receptor subtypes in the transduction of the melatonin signal. A recent study with mt1 receptor-deficient mice has suggested that the two melatonin effects on the mouse suprachiasmatic nuclei, inhibition of neuronal firing and entrainment of the circadian rhythm, could be independently mediated by the two receptor subtypes (Liu et al., 1998). The study of the different roles of the two receptor subtypes is further impeded by the lack of selective melatonin receptor agonists. Some melatonin receptor antagonists have been reported to be highly selective for the human MT2 receptor subtype (Dubocovich et al., 1997), but their efficacy has not yet been tested at the human melatonin receptor subtypes. For this reason we determined the affinity and efficacy of one of these analogues (4P-PDOT) at both human mt1 and MT2 receptors in the present study. Recently we have studied the affinity and efficacy of a number of known melatonin analogues at the mt1 human melatonin receptor stably expressed in NIH3T3 fibroblast cells, using the agonist-mediated [35S]-GTPγS binding method for evaluation of the efficacy (Nonno et al., 1998). Agonist stimulation of G protein-coupled receptors induces conformational changes in their cognate G proteins, leading to activation of the G protein and effector activation. The essential step for G protein activation is the GDP/GTP exchange (Birnbaumer et al., 1990), that can be quantified by the use of [35S]-GTPγS (Hilf et al., 1989; Lorenzen et al., 1993). The amount of [35S]-GTPγS incorporation into membranes at maximal concentrations of agonist is related to the intrinsic activity of the agonist itself. Studies on the efficacy of melatonin analogues at the human MT2 receptor are still lacking. Here we report the pharmacological characterization of the MT2 receptor stably expressed in NIH3T3 mouse fibroblast cells and the activity of known melatonin agonists, partial agonists and antagonists.

The synthesis of several novel indole melatonin analogues substituted at the 2-position with acylaminomethyl (8-11), acylaminoethyl (5a-k), or acylaminopropyl (13) side chains is reported. On the basis of a novel in vitro functional assay (specific binding of [35S]GTPgammaS), which can discriminate agonist from partial agonist, antagonist, and inverse agonist ligands, 5a,g, h,j and 13 were shown to be partial agonists, 5d,e and 8-11 competitive antagonists, and 5b,c,k putative inverse agonists. Binding and functional assays were performed on cloned human MT1 receptor. Structure-activity relationship considerations indicate that N-[1-aryl-2-(4-methoxy-1H-indol-2-yl)(C1-C2)alkyl]alkanamides represent a lead structure for this type of ligands.

Mouse fibroblasts (NIH3T3) transfected with the full-length coding region of the Mel1a melatonin receptor stably expressed the receptor, coupled to a pertussis toxin-sensitive G-protein(s) and exhibiting high affinity and adequate pharmacological profile. The receptor protein had the tendency of a strong coupling to the G-protein and therefore low-affinity state was induced by uncoupling the receptor from its G-protein in presence of high concentrations of NaCl (500–700 mM) and/or GTPγS (100 μM). Thereafter, the affinity of a series of melatonin analogues was determined to both, high- and low-affinity receptor states, thus providing a basis for the prediction of their efficacy, according to the ternary complex model. The cells were subsequently used to study the agonist-induced G-protein activation, determined by calculating the rate of GDP-GTP exchange measured in presence of 35S-labelled GTPγS. The natural ligand melatonin induced a significant increase in the GDP-GTP exchange rate, the presence of GDP and NaCl being necessary to observe this effect. The full agonists 2-phenylmelatonin, 2-bromomelatonin and 6-chloromelatonin equally induced an increase of the GDP-GTP exchange. 5-Hydroxy-N-acetyltryptamine activated the GTP-GDP exchange to a much lesser extent (53%) than melatonin, thus behaving as a partial agonist. As predicted by the model, the melatonin antagonist (N-[(2-phenyl-1H-indol-3-yl)ethyl]cyclobutanecarboxamide) was without effect on basal G protein activation. Coincubation of this compound with melatonin induced a dose-dependent rightward shift in the melatonin concentration-effect curve, thus exhibiting the behaviour of a competitive and surmountable antagonist. Using the equation proposed by Venter (1997) we were able to determine that there were no ‘spare’ receptors in the system. Therefore, the approach proposed in the present work can be successfully used for the determination of ‘drug action’ at the level of the human Mel1a melatonin receptor and evaluation of the efficacy of new selective melatonin analogues.

Asthma is associated with structural remodelling processes, including basement membrane thickening, increased vascularity and smooth muscle alterations. It is known that respiratory infections are associated with asthma exacerbation; infections can worsen asthma symptoms and influence susceptibility to asthma onset. How infections affect asthma is not fully elucidated. It is possible that the immune response, due to recurrent infections, leads to the pathogen's eradication but also increases bronchial inflammation, which induces airway remodelling in asthmatic subjects. We evaluated how infection affects lung remodelling and inflammatory responses and assessed the impact of antibiotic treatment in a murine model of asthma. Ovalbumin-sensitised BALB/c mice were divided into control, mild and chronic asthmatics. A subset of animals in each group was infected with Streptococcus pneumoniae and was treated with antibiotics. The results show an increase in key lung remodelling factors in mice with chronic asthma, particularly those infected with S. pneumoniae. Notably, antibiotic therapy attenuated these effects. These findings demonstrate for the first time that prompt antibiotic therapy may be useful to reduce lung remodelling progression in infected asthmatic subjects.

The adult human cerebellum expresses melatonin receptors with high density in the external zone of the molecular layer. Cloning of the receptor cDNA isolated by RT-PCR from human cerebellar specimens and sequencing analysis of the full-length coding region revealed that the receptor protein is encoded by a transcript identical to that recently cloned from the human hypothalamus (Mel1a). In situ hybridization using an antisense cRNA-probe demonstrated that the melatonin receptor mRNA is localized in the cerebellar granule cells. Mapping of the messenger by RT-PCR with Mel1a specific primers in different areas of the human brain disclosed a quite widespread distribution of the transcript, although expressed at very low levels. Semi-quantitative comparison between the different brain regions allowed to establish the following relative mRNA abundance: cerebellum > or = occipital cortex > or = parietal cortex > temporal cortex > thalamus > frontal cortex > or = hippocampus. No mRNA was detected in white blood cells.

The aim of the study was to examine the In vitro susceptibility of clinical isolates of respiratory pathogens to clofoctol compared with amoxicillin and erythromycin, and to characterize the pharmacokinetic/pharmacodynamic (PK/PD) relationships of clofoctol using a murine pneumonia infection model. Strains clinically isolated from patients between 2005 and 2009 were used to examine susceptibility: penicillin-susceptible Streptococcus pneumoniae, penicillin-resistant S. pneumoniae, Streptococcus pyogenes, methicillin-susceptible Staphylococcus aureus, methicillin-resistant S. aureus, and Haemophilus influenzae. The In vitro activity of clofoctol against clinical isolates has essentially remained unchanged over recent years. The MIC₅₀ and MIC₉₀ of clofoctol against penicillin-resistant S. pneumoniae are lower than that of amoxicillin and erythromycin. The area under curve/minimum inhibitory concentration (AUC/MIC) ratio is the PK/PD parameter that best correlates with in vivo clofoctol efficacy; the value of AUC/MIC required to achieve the maximum effect in this study was 75.5.

The design, synthesis, and biological profile of several indole melatonin analogues with a conformationally restricted C3 amidoethane side chain are presented. Examination of the accessible conformations of the melatonin side chain led us to explore some of its fully or partially restricted analogues, 2−12, the binding affinity values of which were utilized to gain further insight on the melatonin binding site. Two pharmacophoric models have been devised for melatonin and the active compounds by conformational analysis and superimposition performed using the DISCO program. In these models, the melatonin side chain can adopt a gauche/anti conformation out of the indole plane. Another contribution of this study regards the observation of a possible binding point interaction around the C2 position of the indole, as suggested by the remarkably increased binding affinity observed in the C2-substituted analogues 6 and 9 and especially in the more rigid analogue 5. The biological activity and the efficacy of the...

A series of phenoxyalkyl and phenylthioalkyl amides were prepared as melatoninergic ligands. Modulation of affinity of the newly synthesized compound by applying SARs around the terminal amide moiety, the alkyl chain, and the methoxy group on the aromatic ring provides compounds with nanomolar affinity for both melatonin receptor subtypes. Affinity towards MT(1) and MT(2) receptors were modulated also exploiting chirality. The investigation of intrinsic activity revealed that all the tested compounds behave as full or partial agonists.

The class of N-(anilinoethyl)amides includes melatonin receptor ligands with varied subtype selectivity and intrinsic activity. One of these ligands, the MT(2)-selective partial agonist UCM765 (N-{2-[(3-methoxyphenyl)phenylamino]ethyl}acetamide), had evidenced hypnotic effects in rodents at doses > or =40 mg kg(-1) (s.c.), in spite of its sub-nanomolar affinity for human melatonin receptors. Supposing that its low in vivo potency could be due, at least in part, to metabolic liability in rat liver, UCM765 was incubated with rat liver S9 fraction and rat, mouse, or human microsomes, and the major metabolites were identified by LC-MS, synthesized, and in vitro tested for their affinity toward MT(1) and MT(2) receptors. The obtained information was exploited to design novel analogues of UCM765 that are more resistant to in vitro oxidative degradation, while maintaining a similar binding profile. The analogue UCM924 (N-{2-[(3-bromophenyl)-(4-fluorophenyl)amino]ethyl}acetamide) displayed a binding profile similar to that of UCM765 on cloned human receptors (MT(2)-selective partial agonist) and a significantly longer half-life in the presence of rat liver S9 fraction.

Serenoa repens extract is the phytotherapeutic agent most frequently used for the treatment of the urological symptoms caused by benign prostatic hyperplasia. There are many extracts in the market and each manufacturer uses different extraction processes; for this reason, it's possible that one product is not equivalent to another. The aim of this study was to compare the activity of different extracts of Serenoa repens marketed in Italy.The following extracts were tested on 10 day co-cultured epithelial and fibroblast cells by a 5alpha-reductase activity assay: Permixon, Saba, Serpens, Idiprost, Prostamev, Profluss and Prostil. In order to assess the variability in Serenoa repens products, 2 different batches for each brand were evaluated.All extracts tested, albeit variably, are able to inhibit both isoforms of 5alpha-reductase. However, the potency of the extracts appears to be very different, as well as the potencies of 2 different batches of the same extract. This is probably due to qualitative and quantitative differences in the active ingredients. So, the product of each company must be tested to evaluate the clinical efficacy and bioactivity.

A novel series of melatonin receptor ligands, characterized by a N-(substituted-anilinoethyl)amido scaffold, along with preliminary structure-activity relationships (SARs), is presented. MT1 and MT2 receptor binding affinity and intrinsic activity have been modulated by the introduction of different substituents on the aniline nitrogen, on the benzene ring, and on the amide side chain. Modulation of intrinsic activity and MT2 selectivity of the newly synthesized compounds has been achieved by applying SAR models previously developed, providing compounds with different binding and intrinsic activity profiles. Compound 3d, with a bulky ss-naphthyl group, behaves as an MT2-selective antagonist with sub-nM affinity. Size reduction of the substituent enhances intrinsic activity, as in the nonselective N-methyl-anilino agonist 3i. The phenyl derivative 3g is an MT2-selective partial agonist, with MT2 binding affinity higher than melatonin, showing promising sleep-promoting and antianxiety properties in animal models.

Racemic N-(8-methoxy-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-10-ylmethyl)acetamide (compound 5) was previously identified as a novel selective MT(2) antagonist fulfilling the requirements of pharmacophore and 3D QSAR models. In this study the enantiomers of 5 were separated by medium-pressure liquid chromatography and behaved as the racemate. Compound 5 was modified at the acylaminomethyl side chain and at position C8. The resulting analogues generally behaved as melatonin receptor antagonists (GTPgammaS test) with a modest degree of selectivity (up to 10-fold) for the MT(2) receptor. Changes at the amide side chain led to a decrease in binding affinity, whereas 8-acetyl and 8-methyl derivatives 12 and 11, respectively, were as potent as the 8-methoxy parent compound 5. Docking experiments with an MT(2) receptor model suggested binding modes consistent with the observed SARs and with the lack of selectivity of the enantiomers of 5.

A new series of melatonin (MLT) dimers were obtained by linking together two melatonin units with a linear alkyl chain through the MLT acetamido group or through a C-2 carboxyalkyl function. The binding properties of these ligands were evaluated in in vivo experiments on cloned human MT1 and MT2 receptors expressed in NIH3T3 rat fibroblast cells. The class of 2-carboxyalkyl dimers was the most interesting one with compounds having good MT1/MT2 nanomolar affinity. The data obtained suggest that the spacer length is crucial for optimal interaction at both receptor subtypes as well as to determine functional activity of the resulting dimers.

The in vitro and in vivo antichlamydial activities of dexamethasone and beclomethasone alone and in combination with an antibiotic were tested. In vitro, dexamethasone and beclomethasone decreased the number of inclusion-forming units versus the control number ( P < 0.001). The combination of glucocorticoids with azithromycin, telithromycin, or levofloxacin was more active than antibiotics used alone ( P < 0.001). The combination, tested in a murine Chlamydophila pneumoniae infection model, produced similar results.

Purpose: We investigated the ability of the combinatorial administration of different inhibitors with activities on glioma angiogenesis, migration, and proliferation to produce a prolonged inhibition of glioma growth. Experimental Design: We combined inhibitors affecting solely tumor angiogenesis (PF-4/CTF, cyclo-VEGI) or inhibitors affecting both angiogenesis and invasion together (PEX, PF-4/DLR). Results: When administered in combination, these drugs produced a prolonged and increased inhibition of glioma growth independently from the type of inhibitor used. The combinatory administration was more effective than the administration of a single inhibitor alone, and a strong therapeutic response was reached with a significantly lower amount of protein. The strongest inhibition was observed when human PEX and PF-4/DLR, which affect both glioma angiogenesis and invasion by separate mechanisms, were combined. Conclusions: This supports the concept that prolonged glioma growth inhibition can be achieved by simultaneous delivery of molecules that target both tumor and endothelial cells and acting by separate mechanisms.

This work reports the design and synthesis of novel alkylamides, characterized by a dibenzo[a,d]cycloheptene nucleus, as melatonin (MLT) receptor ligands. The tricyclic scaffold was chosen on the basis of previous quantitative structure-activity studies on MT1 and MT2 antagonists, relating selective MT2 antagonism to the presence of an aromatic substituent out of the plane of the MLT indole ring. Some dibenzo seven-membered structures were thus selected because of the noncoplanar arrangement of their benzene rings, and an alkylamide chain was introduced to fit the requirements for MLT receptor binding, namely, dibenzocycloheptenes with an acylaminoalkyl side chain at position 10 and dibenzoazepines with this side chain originating from the nitrogen atom bridging the two phenyl rings. Binding affinity at human cloned MT1 and MT2 receptors was measured by 2-[125I]iodomelatonin displacement assay and intrinsic activity by the GTPgammaS test. The majority of the compounds were characterized by higher affinity at the MT2 than at the MT1 receptor and by very low intrinsic activity values, thus confirming the importance of the noncoplanar arrangement of the two aromatic rings for selective MT2 antagonism. Dibenzocycloheptenes generally displayed higher MT1 and MT 2affinity than dibenzoazepines. N-(8-Methoxy-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-10-ylmethyl)propionamide (4c) and -butyramide (4d) were the most selective MT2 receptor antagonists of the series, with MT2 receptor affinity comparable to that of melatonin and as such among the highest reported in the literature for MLT receptor antagonists. The acetamide derivative 4b produced a noticeable reduction of GTPgammaS binding at MT2 receptor, thus being among the few inverse agonists described.

The systemic administration of endogenous inhibitors significantly reduced the growth of human glioma in vivo, but required the production of a large amount of biologically active protein. In this study we reduced the amount of protein needed and optimized the therapeutical response by delivering the endogenous inhibitors locally into the brain by osmotic minipumps. Human hemopexin fragment of MMP-2 or COOH-terminal fragment of platelet factor-4 were delivered locally and continuously into the brain of mice implanted intracranially with glioma cells, by osmotic minipumps connected to an intracranial catheter. Local delivery of human hemopexin fragment of MMP-2 and COOH-terminal fragment of platelet factor-4 significantly inhibited the growth of well-established malignant glioma in nude and BALB/C mice. When the inhibitors were given at the same concentration, the efficacy of the local delivery was much higher than that reached with the systemic administration, both when the inhibitor was administered daily or continuously by s.c. minipumps. Moreover, the local delivery reduced the amount of protein needed to reach a significant therapeutic response. Intracerebral delivery maintained a long-term control of glioma growth and inhibited glioma recurrence in a surgical resection model. Treatment showed no side effects. Histochemical analysis of tumors showed that the tumor growth inhibition was the result of a decrease in tumor vasculature and a change in tumor vessel morphology. Our data demonstrate that local intracerebral delivery of endogenous inhibitors effectively inhibits malignant glioma growth and reduces the amount of protein needed to reach a therapeutical response.

The design, synthesis, and biological profile of several indole melatonin analogues with a conformationally restricted C3 amidoethane side chain are presented. Examination of the accessible conformations of the melatonin side chain led us to explore some of its fully or partially restricted analogues, 2-12, the binding affinity values of which were utilized to gain further insight on the melatonin binding site. Two pharmacophoric models have been devised for melatonin and the active compounds by conformational analysis and superimposition performed using the DISCO program. In these models, the melatonin side chain can adopt a gauche/anti conformation out of the indole plane. Another contribution of this study regards the observation of a possible binding point interaction around the C2 position of the indole, as suggested by the remarkably increased binding affinity observed in the C2-substituted analogues 6 and 9 and especially in the more rigid analogue 5. The biological activity and the efficacy of the new compounds were tested by measuring the inhibition of the forskolin-stimulated cAMP accumulation and the GTP gamma S index. Both analyses demonstrated that all of the compounds were full agonists with the exception of 4 and 9, which showed a slight reduction in efficacy and would seem to be partial agonists.

A new series of indole melatonin analogues, bearing the amido ethyl side chain attached at the N-1 position of the indole nucleus, were synthesized and tested for their affinity for the melatonin receptor isolated from quail optic tecta in a series of in vitro ligand-binding experiments using 2-[125I]iodomelatonin as the labeled ligand. The biological activity was evaluated using two models: effects on the forskolin-stimulated cAMP accumulation in explants from quail optic tecta and evaluation of the GTP gamma S index derived from competition experiments performed in the absence or presence of GTP gamma S. Compounds 2a and 2k-n, obtained by shifting the methoxy group and the ethylamido side chain from the C-5 and C-3 positions of melatonin to the C-6 and N-1 positions of the indole nucleus, exhibited an affinity similar to that of melatonin itself, as well as full agonist activity. Optimization of the C-2 substituent by introducing Br, phenyl, or COOCH3 (2b-d) resulted in a significantly enhanced affinity (in the picomolar range) and improved agonist biological activity. Compounds lacking the methoxy group and bearing an N-alicyclic group (2h-j) behaved as partial agonists or antagonists.