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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.

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.

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.

Glioma recurrences develop at the borders of the surgical cavity and are the main cause of their poor prognosis. There are no therapeutic advances to reduce the incidence of recurrence or animal models that closely mimic the clinical scenario to evaluate novel therapeutics. This work investigates the efficacy of endogenous inhibitors, in preventing the recurrence of human malignant gliomas, in a newly developed animal model of glioma surgical resection. We developed a nude mice model in which human glioma xenografts were microsurgically removed. After surgery, small islets of tumor cells persisted in the normal brain parenchyma, grew, and formed a recurrence. As inhibitors we used PEX and a fragment of platelet factor 4 (PF-4/CTF), which were administered systemically on a daily basis or in metronomic combination with chemotherapy for 120 days. Treatment was started 1 or 15 days after tumor removal. PEX or PF-4/CTF produced a significant improvement in survival, and delayed the appearance of glioma recurrence. Survival of animals that received daily PEX or PF-4/CTF was similar to that of animals that received metronomic PEX or PF-4/CTF and chemotherapy, respectively. The effect of treatment was dependent on the time at which the treatment was initiated. The highest level of inhibition was observed when the treatment was administered 1 day after surgical resection and when PEX was used as the inhibitor (120 days versus 35 days of the control). Tumors treated with PEX or PF-4/CTF were small and well delineated, with few vessels. Postsurgical administration of PEX or PF-4/CTF significantly reduces the incidence human malignant glioma recurrences for a long period of time.

The growth of gliomas depends on the balance of factors stimulating or inhibiting angiogenesis, tumor cell invasion and proliferation. The administration of endogenous inhibitors to experimental human gliomas in animal models resulted in a significant inhibition of tumor growth. It is becoming apparent that resistance can develop over time to many types of endogenous inhibitors and seems to be influenced by the tumor type and system of delivery.We recently isolated a potent endogenous inhibitor, called human PEX, from human glioma cells in culture. Human PEX is a potent inhibitor of angiogenesis, tumor and endothelial cell proliferation and migration. In this paper, we investigated the ability of human PEX to sustain inhibition of glioma growth for a prolonged period of time. We initially developed a recombinant form of the inhibitor and showed that this form had similar in vitro and in vivo activities to the natural one. Human PEX was then administered to nude mice intracranial human glioma model, in combination with metronomic chemotherapy, for a period of 185 days, starting 15 days after tumor cells implantation.Our data showed that the systemic administration of human PEX mantained a very prolonged inhibition of glioma growth (50% survival of animals treated with 2 mg/kg/days was 160 days vs 24 days of the control) and had a synergistic effect with low dose chemotherapy. Histological analysis of tumors, showed that treatment with PEX was associated with a decrease of vascularity, cell proliferation, and increase in apoptosis.These data indicate that human PEX controls tumor growth by separate mechanisms. In addition, treatment with PEX produced well delineated tumors, indicating the persistence of a direct anti-invasive effect of the molecule even after a prolonged period of treatment.

We have studied fasting plasma tryptophan (TRP) levels and tryptophan/large neutral amino acid (TRP/LNAA) ratios in 12 patients with obsessive-compulsive disorder (OCD) and 12 patients with OCD and a coexisting current diagnosis of major depressive disorder (OCD-MDD). Assessments were made at baseline and after 6 weeks of treatment with fluvoxamine. OCD-MDD patients had significantly lower baseline TRP levels and TRP/LNAA ratios than OCD patients. After 6 weeks of fluvoxamine treatment, OCD-MDD patients had significant increases in plasma TRP and TRP/LNAA ratio, whereas OCD patients had non-significant decreases. Our data suggest that a major depressive syndrome could be a state variable affecting the changes in plasma TRP and TRP/LNAA ratio in OCD patients. (C) 1997 Elsevier Science Ireland Ltd. Z8 0 ZR 0 ZS 0 ZB 8

A practical synthesis of N-[2-(2-bromo-5-methoxy-1H-indol-3-yl)ethyl]- acetamide (2-bromomelatonin) was achieved by direct bromination of melatonin with N-bromosuccinimide (NBS) in anhydrous acetic acid at room temperature under nitrogen, followed by flash-chromatography. 1H-NMR and mass spectra showed the bromine to be incorporated at the C-2 position of the indole moiety. Tests performed in vitro with isolated melatonin receptors from rabbit parietal cortex demonstrated that the relative binding affinity of 2-bromomelatonin was about ten times higher than that of melatonin and close to that of 2-iodomelatonin. 2-Bromomelatonin behaved as a potent agonist in the physiological studies. It showed enhanced activity in inhibiting the spontaneous firing activity of cortical neurons and similarly to melatonin and 2-iodomelatonin potentiated significantly the inhibitory effect of GABA. 2-Bromomelatonin was also an extremely effective agonist in the tests performed in vivo in the Syrian hamster gonadal regression model.

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.

Stankov B, Møller, M, Lucini V, Capsoni S, Fraschini F. A carnivore species (Canis familiaris) expresses circadian melatonin rhythm in the peripheral blood and melatonin receptors in the brain. Eur J Endocrinol 1994;131:191–200. ISSN 0804–4643 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) (GTPΓS) 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 μmol/l, and co-incubation with GTPΓS did not modify the binding density. Collectively, these data show that the dog posesses 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. Bojidar Stankov, Chair of Chemotherapy, Department of Pharmacology, University of Milan, 32 Via Vanvitelli, 20129 Milano, Italy

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.

The distribution and the properties of the melatonin binding sites were characterized in the brain of the rabbit by combined use of autoradiography and in vitro ligand-receptor binding. Autoradiography revealed widespread specific binding in the brain. The pars tuberalis of the pituitary gland, suprachiasmatic nuclei, ventromedial hypothalamic nuclei, tapetum, hippocampus, indusium griseum, cingulate gyrus, cortex and the choroid plexus were intensely labelled. Diffuse specific binding was recorded in the olfactory bulb and the anterior hypothalamus. Series of in vitro ligand-receptor binding experiments, using the anterior hypothalamus, confirmed that the binding was of high affinity and specificity. Coincubation with a non-hydrolyzable GTP analogue provoked a shift in the binding affinity, the numerical values of the K d increasing from 20–30 pM to 280–300 pM. Apparently the melatonin receptor in the rabbit brain is linked to its second messenger via a G protein, similarly to what has been described for the brain of other vertebrates.

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.