Randomized trial of safinamide add-on to levodopa in Parkinson's disease with motor fluctuations.

Levodopa is effective for the motor symptoms of Parkinson's disease (PD), but is associated with motor fluctuations and dyskinesia. Many patients require add-on therapy to improve motor fluctuations without exacerbating dyskinesia. The objective of this Phase III, multicenter, double-blind, placebo-controlled, parallel-group study was to evaluate the efficacy and safety of safinamide, an α-aminoamide with dopaminergic and nondopaminergic mechanisms, as add-on to l-dopa in the treatment of patients with PD and motor fluctuations. Patients were randomized to oral safinamide 100 mg/day (n = 224), 50 mg/day (n = 223), or placebo (n = 222) for 24 weeks. The primary endpoint was total on time with no or nontroublesome dyskinesia (assessed using the Hauser patient diaries). Secondary endpoints included off time, Unified Parkinson's Disease Rating Scale (UPDRS) Part III (motor) scores, and Clinical Global Impression-Change (CGI-C). At week 24, mean ± SD increases in total on time with no or nontroublesome dyskinesia were 1.36 ± 2.625 hours for safinamide 100 mg/day, 1.37 ± 2.745 hours for safinamide 50 mg/day, and 0.97 ± 2.375 hours for placebo. Least squares means differences in both safinamide groups were significantly higher versus placebo. Improvements in off time, UPDRS Part III, and CGI-C were significantly greater in both safinamide groups versus placebo. There were no significant between-group differences for incidences of treatment-emergent adverse events (TEAEs) or TEAEs leading to discontinuation. The addition of safinamide 50 mg/day or 100 mg/day to l-dopa in patients with PD and motor fluctuations significantly increased total on time with no or nontroublesome dyskinesia, decreased off time, and improved parkinsonism, indicating that safinamide improves motor symptoms and parkinsonism without worsening dyskinesia.

Long-term efficacy and safety of safinamide as add-on therapy in early Parkinson's disease.

BACKGROUND AND PURPOSE: Safinamide is an α-aminoamide with both dopaminergic and non-dopaminergic mechanisms of action in Phase III clinical development as a once-daily add-on to dopamine agonist (DA) therapy for early Parkinson's disease (PD). METHODS: Study 017 was a 12-month, randomized, double-blind, placebo-controlled pre-planned extension study to the previously reported Study 015. Patients received safinamide 100 or 200 mg/day or placebo added to a single DA in early PD. The primary efficacy endpoint was the time from baseline (Study 015 randomization) to 'intervention', defined as increase in DA dose; addition of another DA, levodopa or other PD treatment; or discontinuation due to lack of efficacy. Safinamide groups were pooled for the primary efficacy endpoint analysis; post hoc analyses were performed on each separate dose group. RESULTS: Of the 269 patients randomized in Study 015, 227 (84%) enrolled in Study 017 and 187/227 (82%) patients completed the extension study. Median time to intervention was 559 and 466 days in the pooled safinamide and placebo groups, respectively (log-rank test; P = 0.3342). In post hoc analyses, patients receiving safinamide 100 mg/day experienced a significantly lower rate of intervention compared with placebo (25% vs. 51%, respectively) and a delay in median time to intervention of 9 days (P < 0.05; 240- to 540-day analysis). CONCLUSIONS: The pooled data from the safinamide groups failed to reach statistical significance for the primary endpoint of median time from baseline to additional drug intervention. Post hoc analyses indicate that safinamide 100 mg/day may be effective as add-on treatment to DA in PD.

Comparison of the potency of 10 different brands of Serenoa repens extracts.

BACKGROUND: The extract of Serenoa repens is the phytopharmaceutical product most often used for the treatment of urological symptoms associated with benign prostatic hyperplasia (BPH). Several extracts are commercially available but extraction processes vary between manufacturers and thus not all these products are equivalent in terms of active ingredient content and composition of preparations. AIM: As there is a paucity of comparative studies, we compared the activity of different extracts of Serenoa repens widely available on the world market. MATERIALS AND METHODS: Beltrax Uno, Permicaps, Permixon, Prostadyn, Prostagutt, Prostamen, Prostamol Uno, ProstaX, Urocaps and Urogutt were assayed for 5-alpha-reductase activity on 10 day fibroblasts and epithelial cells cocultures. Human fibroblast growth factor (hFGF)-induced-proliferation inhibition was also assayed. RESULTS: As to extract activity, differences were observed between the tested extracts, but all were able to inhibit 5-a-reductase types I and II isoenzymes (5alphaR-I and 5alphaR-II) as well as fibroblast proliferation. CONCLUSIONS: Extract potency differs between products and so does proliferation inhibition potency. Quantitative and qualitative variations in the active ingredient are likely to account for these differences.

Feedback dose alteration significantly affects probability of pathogen eradication in nosocomial pneumonia.

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<0.0001), the presence of combination therapy (p = 0.0014) and whether both MIC and drug concentration(s) were measured (p = 0.0002) significantly affected the probability of a good outcome. For microbiological outcome, the MIC for the beta-lactams (

Simultaneous inhibition of glioma angiogenesis, cell proliferation, and invasion by a naturally occurring fragment of human metalloproteinase-2.

Angiogenesis, tumor cell proliferation, and migration are the hallmarks of solid tumors, such as gliomas. This study demonstrates that a fragment derived from the autocatalytic digestion of matrix metalloproteinase (MMP)-2, called PEX, acts simultaneously as an inhibitor of glioma angiogenesis, cell proliferation, and migration. PEX is detected in the cultured medium of various human glioma, endothelial, breast, and prostate carcinoma cell lines. PEX is purified from the medium of glioma cell lines by chromatography, where PEX is constitutively expressed as a free and a TIMP-2-bound form. In human glioma tissue, PEX expression correlates with histological subtype and grade and with alpha v beta 3 integrin expression to which it is bound. Systemic administration of PEX to s.c. and intracranial human glioma xenografts results in a 99% suppression of tumor growth with no signs of toxicity. Thus, PEX is a very promising candidate for the treatment of human malignant gliomas.

Low-dose chemotherapy combined with an antiangiogenic drug reduces human glioma growth in vivo.

This study evaluates the efficacy of the combination of an antiangiogenic drug and conventional chemotherapeutics for the treatment of experimental human gliomas. As an antiangiogenic, we used recombinant human PEX, a fragment of matrix metalloproteinase-2 that we have previously shown to have a significant antimitotic, anti-invasive, and antiangiogenic properties against human glioblastoma in vitro and in vivo. We used carboplatin and etoposide as the two chemotherapeutic drugs routinely used in our institution (Ospedale Maggiore de Milano) for the treatment of malignant gliomas. Conventional chemotherapeutic drugs were administered at high dose or at a low and semicontinuous regimen. Combined treatment of high-dose chemotherapy and PEX did not produce an improvement of survival in comparison with chemotherapy alone, but it was associated with a decrease in tumor volume, vascularity, and proliferative index and an increased apoptosis. All of these animals experienced severe side effects. The longest survival was documented in animals submitted to low and semicontinuous chemotherapy and antiangiogenic treatment. This regimen was associated with no side effects, marked decrease in tumor volume, vascularity, and proliferative index, and an increased apoptosis. Our data suggest that low-dose chemotherapy in combination with PEX can be successfully used against human malignant glioma in vivo.

Different physiological and behavioural effects of e-cigarette vapour and cigarette smoke in mice.

Nicotine is the primary addictive substance in tobacco smoke and electronic cigarette (e-cig) vapour. Methodological limitations have made it difficult to compare the role of the nicotine and non-nicotine constituents of tobacco smoke. The aim of this study was to compare the effects of traditional cigarette smoke and e-cig vapour containing the same amount of nicotine in male BALB/c mice exposed to the smoke of 21 cigarettes or e-cig vapour containing 16.8 mg of nicotine delivered by means of a mechanical ventilator for three 30-min sessions/day for seven weeks. One hour after the last session, half of the animals were sacrificed for neurochemical analysis, and the others underwent mecamylamine-precipitated or spontaneous withdrawal for the purposes of behavioural analysis. Chronic intermittent non-contingent, second-hand exposure to cigarette smoke or e-cig vapour led to similar brain cotinine and nicotine levels, similar urine cotinine levels and the similar up-regulation of α4ß2 nicotinic acetylcholine receptors in different brain areas, but had different effects on body weight, food intake, and the signs of mecamylamine-precipitated and spontaneous withdrawal episodic memory and emotional responses. The findings of this study demonstrate for the first time that e-cig vapour induces addiction-related neurochemical, physiological and behavioural alterations. The fact that inhaled cigarette smoke and e-cig vapour have partially different dependence-related effects indicates that compounds other than nicotine contribute to tobacco dependence.

Cytosolic androgen receptors in the neuroendocrine tissues of the golden hamster: influence of photoperiod and melatonin treatment.

Hamsters exposed for eight weeks to short photoperiod (LD 10:14) or treated with melatonin in the late afternoon under long photoperiod (LD 14:10) had significantly higher number of cytosolic androgen receptors in the pituitaries, hypothalami and harderian glands, as compared to the long photoperiod (LD 14:10) exposed controls. The numerical value of the apparent Kd was two to three times lower in the hypothalami and pituitaries, but not in the harderian glands of the animals from these groups. These results indicate that alterations in receptor numbers and affinity constants may be responsible for the dramatic changes in the sensitivity of the hypothalamo-pituitary axis to the negative feedback actions of the gonadal steroids, observed under inhibitory photoperiods and that this effect could be duplicated by late afternoon melatonin treatment.

Melatonin signal transduction and mechanism of action in the central nervous system: using the rabbit cortex as a model.

The cortex of the rabbit (Oryctolagus cuniculus) is rich in melatonin binding sites, and particularly abundant is the parietal cortex. Consequently, we characterized the putative melatonin receptor in the parietal cortex by a series of in vitro ligand-receptor binding experiments and biochemical and electrophysiological studies. The in vitro saturation and competition experiments demonstrated that the binding in the crude cortical membrane preparations was of high affinity and specificity. Guanine nucleotides (GDP, GTP, and GTP gamma S) inhibited the specific 2-[125I]iodomelatonin binding in a dose-dependent manner. Coincubation with a nonhydrolyzable GTP analog provoked a shift in the binding affinity; the numerical values of the Kd increased from 20-30 to 200-600 pM. Melatonin, in nanomolar concentrations, was able to inhibit the forskolin-stimulated accumulation of cAMP in parietal cortex explants, and preincubation with pertussis toxin counteracted this effect of melatonin. Apparently, the melatonin binding site in the rabbit parietal cortex is linked to its second messenger via a pertussis toxin-sensitive G-protein, probably of the inhibitory Gi class, similar to what has been described for different parts of the brain of other vertebrates. The experiments on the spontaneous firing activity of single neurons in the third to fourth layer of the parietal cortex in anesthetized animals showed that melatonin and its potent agonist 2-iodomelatonin exhibited gamma-aminobutyric acid (GABA)-like effects and were able alone, in nanomolar concentrations, to significantly slow the neuronal firing activity. Moreover, both melatonin and 2-iodomelatonin potentiated the effect of GABA on the neuronal activity, leading to powerful inhibition of the tested neurons. Undoubtedly, the binding site in the rabbit parietal cortex possesses all of the characteristics of a functional receptor. We suggest that melatonin is involved in the control of fundamental cortical functions and that it acts in concert with GABA, one of the two major inhibitory neurotransmitters in the central nervous system.

2-Substituted 5-methoxy-N-acyltryptamines: synthesis, binding affinity for the melatonin receptor, and evaluation of the biological activity.

A series of 2-substituted 5-methoxy-N-acyltryptamines was synthesized and their affinity for the melatonin receptor, isolated from whole quail brains, was tested in a succession of in vitro ligand-receptor binding experiments, using 2-[125I]iodomelatonin as a labeled ligand. Optimization of the C2 substituent and the N-acyl group resulted in compounds having picomolar affinity for the receptor (vs nanomolar affinity for melatonin). In two tests for evaluation of the biological activity (effects on the spontaneous firing activity of single neurons in the rabbit parietal cortex in situ, and the Syrian hamster gonadal regression model in vivo) most of the analogs behaved as agonists. Isopropyl substitution at C2 alone, or concomitantly with cyclopropyl substitution at the N-acyl position, resulted in much lower affinity and weaker biological effect, or lack of activity in the latter case. Of interest are the compounds 4d (R = phenyl, R1 = CH3) and 4g (R = phenyl, R1 = cyclopropyl), which expressed high affinity for the receptor and apparent antagonistic activity under the conditions of the experimental models employed, though the analog 4g (R = phenyl, (R1 = cyclopropyl) seemingly was a weak antagonist and in situ expressed mixed activity in the higher concentration range. Cyclopropyl substitution at the N-acyl position inevitably resulted in lower affinity for the receptor and weaker biological activity. These data demonstrate that the N-acetyl group is important for both affinity and agonist biological activity. The substituents at C2 are crucial for the affinity of the compound for the receptor and can be utilized to create putative high-affinity agonists or antagonists.

1-(2-Alkanamidoethyl)-6-methoxyindole derivatives: a new class of potent indole melatonin analogues.

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.

2-N-acylaminoalkylindoles: design and quantitative structure-activity relationship studies leading to MT2-selective melatonin antagonists.

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.

Conformationally restrained melatonin analogues: synthesis, binding affinity for the melatonin receptor, evaluation of the biological activity, and molecular modeling study.

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.

2-[N-Acylamino(C1-C3)alkyl]indoles as MT1 melatonin receptor partial agonists, antagonists, and putative inverse agonists.

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.

Melatonin receptor ligands: synthesis of new melatonin derivatives and comprehensive comparative molecular field analysis (CoMFA) study.

The CoMFA methodology was applied to melatonin receptor ligands in order to establish quantitative structure-affinity relationships. One hundred thirty-three compounds were considered: they were either collected from literature or newly synthesized in order to gain information about the less explored positions. To this end, various melatonin derivatives were prepared and their affinity for quail optic tecta melatonin receptor was tested. Compounds were aligned on the putative active conformation of melatonin proposed by our previously reported pharmacophore search, and their relative affinities were calculated from the displacement of 2-[125I]-iodomelatonin on different tissues expressing aMT receptors. Compounds were grouped into three sets according to their topology. Subset A: melatonin-like compounds; subset B: N-acyl-2-amino-8-methoxytetralins and related compounds; subset C:N-acyl-phenylalkylamines and related compounds. CoMFA models were derived for each set, using the steric, electrostatic, and lipophilic fields as structural descriptors; the PLS analyses were characterized by good statistical parameters, taking into account the heterogeneity of the binding data, obtained with different experimental protocols. From the CoMFA model for the melatonin-like compounds, besides the well-known positive effect of 2-substitution, a low steric tolerance for substituents in 1, 6, and 7, and a negative effect of electron-rich 4-substituents were observed; the information provided by the newly synthesized compounds was essential for these results. Moreover, a comprehensive model for the 133 compounds, accounting for a common alignment and a common mode of interaction at the melatonin receptor, was derived (Q2 = 0.769, R2 = 0.905). This model validates our previously reported pharmacophore search and offers a clear depiction of the structure-affinity relationships for the melatonin receptor ligands.

Autoradiographic localization of putative melatonin receptors in the brains of two Old World primates: Cercopithecus aethiops and Papio ursinus.

The distribution of putative melatonin receptors in the brains of two Old World primates of the superfamily Catarrhina, Cercopithecus aethiops and Papio ursinus, was characterized using 2-[125I]iodomelatonin autoradiography. The specific binding demonstrated a discrete distribution pattern. The median eminence was intensely labelled, and examination at the light microscopic level demonstrated that the binding was confined to the small layer of cells comprising the pars tuberalis of the pituitary gland. The collar of pars distalis, present in the baboon (Papio ursinus), was diffusely labelled. No binding was detected in the pars distalis proper or the neural lobe of the pituitary gland. The binding in the suprachiasmatic nuclei was weaker, but well discernible. Diffuse faint specific binding was found in the frontal cortex and the dentate gyrus of the hippocampus. Two non-neural sites expressed strong, well-delineated binding: the walls of some brain blood vessels (the vertebral and spinal arteries, the inferior cerebellar and acoustic arteries, the basilar, pericallosal, internal carotid arteries, the arteries forming the circle of Willis) and the choroid plexuses. Binding in the arteries of the circle of Willis, the pars tuberalis and the suprachiasmatic nuclei was readily displaceable. Addition of 1 microM unlabelled 2-iodomelatonin following 45 min of preincubation with the radioactive ligand completely abrogated the binding. Co-incubation with guanosine 5'-O-(3-thiotriphosphate) led to a significant decrease in the apparent binding density in the pars tuberalis and abolished binding in the suprachiasmatic nuclei, but was without effect on the binding in the walls of the adjacent arteries, forming the circle of Willis, in the cortex and in the hippocampus. This qualitative distribution pattern demonstrates that in the two primate species studied, melatonin high-affinity, G-protein-linked binding sites are present in the pars tuberalis and the hypothalamic suprachiasmatic nuclei, and that melatonin may be acting as a synchronizer of the endogenous pacemakers' circadian activity, apart from its possible reproductive effects at the level of pars tuberalis, where the highest receptor density was observed. The strongly labelled arterial walls, and the flimsy labelled cortex and hippocampus, expressed different characteristics: though the binding was readily reversible, it was apparently not regulated by a guanine nucleotide-binding protein.

Pharmacological characterization of the human melatonin Mel1a receptor following stable transfection into NIH3T3 cells.

1. 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. 2. 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 GTPgammaS (100 microM). 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. 3. 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 GTPgammaS. 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. 4. 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. 5. 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.

Ligand efficacy and potency at recombinant human MT2 melatonin receptors: evidence for agonist activity of some mt1-antagonists.

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 (K(I) = 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 > or = melatonin > luzindole > N-acetyl-tryptamine > or = N-[(2-phenyl-1H-indol-3-yl)ethyl]cyclobutanecarboxamide (compound 6) > N-acetylserotonin. 4P-PDOT exhibited a very high selectivity (approximately 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]-GTPgammaS into isolated membranes via receptor catalyzed exchange of [35S]-GTPgammaS for GDP. The relative intrinsic activity and potency of the compounds were subsequently studied by using [35S]-GTPgammaS incorporation. The order of potency was equal to the order of apparent affinity. Melatonin and full agonists increased [35S]-GTPgammaS binding by 250% over basal (taken as 100%). Luzindole did not increase basal [35S]-GTPgammaS binding but competitively inhibited melatonin-stimulated [35S]-GTPgammaS 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.

Effect of photoperiod and gonadectomy on testosterone metabolism in vitro by the prostate of the golden hamster.

Testosterone metabolism was studied in vitro in the prostate of intact and castrated golden hamsters maintained either in short days (8 h light: 16 h darkness, 8L: 16D) or in long days (14L: 10D). Testosterone was found to be converted into 17 beta-hydroxy-5 alpha-androstan-3-one (5 alpha-DHT), 5 alpha-androstane-3 alpha, 17 beta-diol, 5 alpha-androstane-3, 17-dione and androstenedione. The mean conversion of testosterone to 5 alpha-DHT was higher in prostates from animals maintained in long days than in short days (P less than 0.0025) while that to androstenedione was higher in short days (P less than 0.0005); no significant changes in the formation of the other three metabolites were noted. Castration of animals maintained in short days resulted in a significant (P less than 0.05) decrease in the mean conversion to all four metabolites. In contrast, castration of animals kept in a long-day regime caused a significant (P less than 0.01) decrease in the mean formation of 5 alpha-DHT but a significant (P less than 0.05) increase in the mean formation of 5 alpha-androstane-3 alpha, 17 beta-diol.

A carnivore species (Canis familiaris) expresses circadian melatonin rhythm in the peripheral blood and melatonin receptors in the brain.

Dogs kept under controlled photoperiodic conditions of 12 h light and 12 h dark expressed a clear diurnal melatonin rhythm in the peripheral blood, with a swift peak restricted to the late part of the scotophase. The highest density of high-affinity, G-protein-linked 2-[125I]iodomelatonin binding sites was found in the pars tuberalis of the pituitary gland. Binding sites were found also in the pars distalis, and light microscopy/high-resolution autoradiography showed that binding was located exclusively over the chromophobe and basophilic cells forming the adenopituitary zona tuberalis, well developed in this species, and extending into the gland as a continuation of pars tuberalis. Cords of basophilic cells located in the pars distalis proper also expressed high receptor density. The eosinophils in the adenohypophysis and the neural lobe were devoid of binding. Heavily labeled were the external laminar and the mitral cell layers of the olfactory bulbs, but no binding was detected in the filae nervi olfactorii or tractus olfactorius. The hypothalamic suprachiasmatic nuclei were discernible clearly. Quantitative autoradiography inhibition experiments revealed that the apparent melatonin inhibitory constant (IC50) in all those areas was around 0.1 nmol/l, which is a physiologically appropriate value considering the peripheral blood melatonin levels. Co-incubation with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) led to a consequential decrease in the binding density. The specific binding observed in other areas (hippocampus, frontal, parietal, occipital cortex and cerebellum) was rather weak, diffuse and could not be attributed to a particular layer; the apparent IC50 for melatonin was about 1 mumol/l, and co-incubation with GTP gamma S did not modify the binding density. Collectively, these data show that the dog possess all the prerequisites for an efficient network adapted to photoperiodic time measurements. A circadian melatonin signal in the peripheral blood and an apparently functional readout receptor system located in key positions within the brain are both present in this species.