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Background: According to the views of psychoneuroendocrinoimmunology, many interactions exist between nervous, endocrine and immune system the purpose of which is to achieve adaptive measures restoring an internal equilibrium (homeostasis) following stress conditions. The center where these interactions converge is the hypothalamus. This is a center of the autonomic nervous system that controls the visceral systems, including the immune system, through both the nervous and neuroendocrine mechanisms. The nervous mechanisms are based on nervous circuits that bidirectionally connect hypothalamic neurons and neurons of the sympathetic and parasympathetic system; the neuroendocrine mechanisms are based on the release by neurosecretory hypothalamic neurons of hormones that target the endocrine cells and on the feedback effects of the hormones secreted by these endocrine cells on the same hypothalamic neurons. Moreover, the hypothalamus is an important subcortical center of the limbic system that controls through nervous and neuroendocrine mechanisms the areas of the cerebral cortex where the psychic functions controlling mood, emotions, anxiety and instinctive behaviors take place. Accordingly, various studies conducted in the last decades have indicated that hypothalamic diseases may be associated with immune and/or psychic disorders. Objective: Various researches have reported that the hypothalamus is controlled by the cerebellum through a feedback nervous circuit, namely the hypothalamocerebellar circuit, which bi-directionally connects regions of the hypothalamus, including the immunoregulatory ones, and related regions of the cerebellum. An objective of the present review was to analyze the anatomical bases of the nervous and neuroendocrine mechanisms for the control of the immune system and, in particular, of the interaction between hypothalamus and cerebellum to achieve the immunoregulatory function. Conclusion: Since the hypothalamus represents the link through which the immune functions may influence the psychic functions and vice versa, the cerebellum, controlling several regions of the hypothalamus, could be considered as a primary player in the regulation of the multiple functional interactions postulated by psychoneuroendocrinoimmunology.

Objective:Recent scientific approaches to cancer patients draw attention to the psychological aspects of the disease and the involvement of their families, who are forced to reorganize themselves in order to manage the patient's illness. Functional responses to a stressful event facilitate open communication between family members and empathy for the patient's children, who need to be involved and informed about the illness in a clear and open fashion. The primary goal of this observational study was to explore the communication styles used by cancer-stricken parents with their children and to identify a correlation with the patient's levels of anxiety and depression and their ability to cope. We also sought to understand whether location, severity, and time from diagnosis influenced communication, coping, anxiety, or depression.Method:From September of 2011 to July of 2015, 151 questionnaires were given to patients who had received at least one course of chemotherapy. The instruments that we employed were the Openness to Discuss Cancer in the Nuclear Family Scale, the Hospital Anxiety and Depression Scale, and the Mini-Mental Adjustment to Cancer Scale. Our sample included patients with children aged from 3 to 18 years. The patients had different types of cancer, mainly gastrointestinal and breast cancer. Their disease was at the metastatic stage in approximately 20% of patients.Results:Our results showed statistically significant correlations between higher levels of anxiety and depression and more closed communication styles. The coping styles “hopelessness/helplessness,” “cognitive avoidance,” and “anxious preoccupation” were associated with a closed communication style that is correlated with higher levels of anxiety and depression. Tumor location, time from diagnosis, and stage of disease did not show statistically significant correlations with anxiety, depression, coping mechanisms, or communication styles.Significance of results:Our study confirmed what has been reported in the literature: high levels of anxiety and depression affect communication among family members. Not surprisingly, the “fighting spirit” coping style engenders open communication.

Background: Pretreated metastatic breast cancer (MBC) remains a formidable challenge with unmet needs both in terms of prolonged survival and quality-of-life-related issues. Methods: We collected data from 27 MBC patients treated with gemcitabine and oxaliplatin (GEMOX) at our institution between June 2009 and April 2015. The patients were heavily pretreated, and all had previously been exposed to anthracyclines and taxanes. Results: We achieved a complete response in 1 patient (4%), a partial response in 7 patients (26%) and stable disease in 12 patients (44%), while 6 patients (22%) experienced progressive disease. The response of 1 patient (4%) could not be evaluated because she interrupted her treatment during the first cycle due to a major reaction to oxaliplatin. We observed grade 4 hypertransaminasaemia in only 1 patient (4%) and grade 2 neuropathy in 16 patients (59%). Grade 3 leuconeutropenia was observed in 5 patients (18%). The median progression-free survival was 5.9 months and the median overall survival was 9.6 months. Conclusions: GEMOX is an efficient and well-tolerated salvage regimen for MBC patients.

Based on their high selectivity of action and low toxicity, naturally occurring peptides have great potential in terms of drug development. However, the pharmacokinetic properties of peptides, in particular their half life, are poor. Among different strategies developed for reducing susceptibility to peptidases, and thus increasing the duration of action of peptides, the generation of branched peptides has been described. However, the synthesis and purification of branched peptides are extremely complicated thus limiting their druggability. Here we present a novel and facile synthesis of tetrabranched peptides acting as GPCR ligands and their in vitro and vivo pharmacological characterization. Tetrabranched derivatives of nociceptin/orphanin FQ (N/OFQ), N/OFQ related peptides, opioid peptides, tachykinins, and neuropeptide S were generated with the strategy named peptide welding technology (PWT) and characterized by high yield and purity of the desired final product. In general, PWT derivatives displayed a pharmacological profile similar to that of the natural sequence in terms of affinity, pharmacological activity, potency, and selectivity of action in vitro. More importantly, in vivo studies demonstrated that PWT peptides are characterized by increased potency associated with long lasting duration of action. In conclusion, PWT derivatives of biologically active peptides can be viewed as innovative pharmacological tools for investigating those conditions and states in which selective and prolonged receptor stimulation promotes beneficial effects.

In the vascular system, angiogenesis and arteriogenesis play a unique yet equally important role in both health and disease. Angiogenesis, the formation of new blood vessels from a preexisting vascular bed, occurs naturally during wound healing, the female menstrual cycle and pregnancy. It plays a critical role in tissue growth and repair, and is a highly controlled process that is dependent on an intricate balance of both pro-angiogenic (to stimulate) and anti-angiogenic (to negatively regulate the phenomenon) factors. Otherwise, the term arteriogenesis refers to anatomic transformation of preexisting arterioles with increasing lumen area and wall thickness, due to a thick muscular layer and purchasing of visco-elastic and vasomotor capacities. Arteriogenesis differs from angiogenesis in several aspects, the most important being the dependence of angiogenesis on hypoxia and the dependence of arteriogenesis on inflammation. The expression of growth factors and the cooperation of surrounding and infiltrating cells seem to be essential in orchestrating the complex processes during arteriogenesis.

The cerebrocerebellar circuit is a feedback circuit that bidirectionally connects the neocortex and the cerebellum. According to the classic view, the cerebrocerebellar circuit is specifically involved in the functional regulation of the motor areas of the neocortex. In recent years, studies carried out in experimental animals by morphological and physiological methods, and in humans by magnetic resonance imaging, have indicated that the cerebrocerebellar circuit is also involved in the functional regulation of the nonmotor areas of the neocortex, including the prefrontal, associative, sensory and limbic areas. Moreover, a second type of cerebrocerebellar circuit, bidirectionally connecting the hypothalamus and the cerebellum, has been detected, being specifically involved in the regulation of the hypothalamic functions. This review analyzes the morphological features of the centers and pathways of the cerebrocerebellar circuits, paying particular attention to their organization in different channels, which separately connect the cerebellum with the motor areas and nonmotor areas of the neocortex, and with the hypothalamus. Actually, a considerable amount of new data have led, and are leading, to profound changes on the views on the anatomy, physiology, and pathophysiology of the cerebrocerebellar circuits, so much they may be now considered to be essential for the functional regulation of many neocortex areas, perhaps all, as well as of the hypothalamus and of the limbic system. Accordingly, clinical studies have pointed out an involvement of the cerebrocerebellar circuits in the pathophysiology of an increasing number of neuropsychiatric disorders.

Nociceptin/Orphanin FQ (N/OFQ) regulates several biological functions via selective activation of the N/OFQ receptor (NOP). In this study novel nonpeptide NOP ligands were characterized in vitro in receptor binding and [35S]GTPγS stimulated binding in membranes of cells expressing human NOP and classical opioid receptors, calcium mobilization assay in cells coexpressing the receptors and chimeric G proteins, bioluminescence resonance energy transfer (BRET) based assay for studying NOP receptor interaction with G protein and arrestin, the electrically stimulated mouse vas deferens and the mouse colon bioassays. The action of the AT compounds were compared with standard NOP agonists (N/OFQ and Ro 65-6570) and the NOP selective antagonist SB-612111. AT compounds displayed high NOP affinity and behaved as NOP agonists in all the functional assays consistently showing the following rank order of potency AT-127≥AT-090≥AT-035 > AT-004= AT-001. AT compounds behaved as NOP full agonists in the calcium mobilization and mouse colon assays and as partial agonists in the [35S]GTPγS and BRET assays. Interestingly AT-090 and AT-127, contrary to standard nonpeptide agonists that display G protein biased agonism, behaved as an unbiased agonists. AT-090 and AT-127 displayed higher NOP selectivity than Ro 65-6570 at native mouse receptors. AT-090 and AT-127 might be useful pharmacological tools for investigating the therapeutic potential of NOP partial agonists.

An innovative chemical strategy named peptide welding technology (PWT) has been developed for the facile synthesis of tetrabranched peptides. [Dmt1]N/OFQ(1-13)-NH2 acts as a universal agonist for nociceptin/orphanin FQ (N/OFQ) and classical opioid receptors. The present study investigated the pharmacological profile of the PWT derivative of [Dmt1]N/OFQ(1-13)NH2 (PWT2-[Dmt1]) in several assays in vitro and in vivo after spinal administration in monkeys subjected to the tail withdrawal assay. PWT2-[Dmt1] mimicked the effects of [Dmt1]N/OFQ(1-13)-NH2 displaying full agonist activity, similar affinity/potency and selectivity at human recombinant N/OFQ (NOP) and opioid receptors in receptor binding, stimulation of [35S]GTPγS binding, calcium mobilization in cells expressing chimeric G proteins, and BRET studies for measuring receptor/G-protein and receptor/β-arrestin 2 interaction. In vivo in monkeys PWT2-[Dmt1] elicited dose-dependent and robust antinociceptive effects being more potent and longer lasting than [Dmt1]N/OFQ(1-13)-NH2. The analgesic action of PWT2-[Dmt1] was sensitive to the NOP receptor antagonist J-113397, but not naltrexone. Thus, the present study demonstrated that the tetrabranched derivative of [Dmt1]N/OFQ(1-13)-NH2 obtained with the PWT technology maintains the in vitro pharmacological profile of the parent peptide but displays higher potency and longer lasting action in vivo.

Mast cells (MCs) are localized in connective tissues and are more numerous near the boundaries between the external environment and the internal milieu including the skin, the respiratory tract, the gastrointestinal tract and the conjunctiva. In the gastrointestinal tract, MCs represent 1-5% of mononuclear cells in the lamina propria of the mucosa and in the submucosa, and they are also found inside the epithelium and deep in the muscle and serosal layers. The gastrointestinal MCs perform their biological functions, releasing mediators, as amines (histamine, serotonin), cytokines, proteases, lipid mediators (leukotrienes, prostaglandins), and heparin. MCs are involved in the pathogenesis of different inflammatory conditions and tumors of the gastrointestinal tract. The use of MCs' tryptase inhibitors or c-KitR tyrosine kinase inhibitors could represent a potential anti-MC therapeutic approach in all the inflammatory and tumor pathological conditions of the digestive tube in which MCs are involved.

e14018 Background: Chemotherapy improves the survival rate of stage III colon cancer patients. The combination of oxaliplatin, 5-fluorouracil and leucovorin (the FOLFOX4 regimen) has emerged as the standard of care. Cognitive changes and disfunction after cancer chemotherapy are increasingly reported as a disturbing side-effect in cancer survivors. This prospective study evaluates potential alterations in cognitive function in Folfox4-treated patients. Methods: We evaluated 57 consecutive colorectal cancer patients who received adjuvant chemotherapy with FOLFOX4. Patients underwent a complete battery of neuropsychological tests at three different times: before (T0), at the end (T1) and six months after treatment (T2). Changes in neuropsychological test scores at T0, T1 and T2 were compared with a two tail t-test (T1-T0, T2-T1, T2-T0), using the repeated measure ANOVA model. Results: We have analyzed cognitive impairment (Mini Mental State Examination, MMSE), visuo-spatial memory, information processing speed (Trial Making Test-A and Trial Making Test-B), verbal memory (Rey Auditory Verbal Learning Test), emotional distress (Psychological Distress Inventory), anxiety (State and Trait Anxiety Inventory) and depression (Beck Depression Inventory). We found no cognitive impairment in time considered, as we found a median MMSE score of 27,51 ± 1,21 at T0, 27,63 ± 0,65 at T1 and 27,26 ± 1,17 at T2. We noticed some transient variations in tests evaluating verbal memory and in information processing speed performances. The only significative scores modifications were those related to anxiety and depression. Conclusions: We found no significant effect on cognitive function related to chemotherapy, the only little modification is about some emotional performance during chemotherapy. These findings may be explained by the central role of the psychological adaptation process, which occurs during the period from diagnosis to completion of treatment and is characterized by anxiety and adjustment depression. Our results seem to rule out any significant cognitive impairment due to adjuvant Folfox4 chemotherapy in colon cancer patients.

Neuropeptide S (NPS), the endogenous ligand of a previously orphan receptor now named NPSR, regulates various biological functions in the brain, including arousal, locomotion, anxiety, and food intake. Here we report on a focused structure-activity study of Gly5, which has been replaced with L and D amino acids. Fifteen NPS related peptides were synthesized and pharmacologically tested for intracellular calcium mobilization using HEK293 cells stably expressing the mouse NPSR. The results of this study demonstrated that peptide potency is inversely related to the side chain size, while peptide efficacy strongly depends on the relative L and D configuration, with the L amino acids favoring agonist while D amino acids display antagonist pharmacological activity. [D-Val5]NPS behaved as NPSR pure antagonist in HEK293(mNPSR) cells showing the highest potency (pK(B) 7.56) among this series of peptides. The antagonist action of [D-Val5]NPS was confirmed in vivo in mice, where the peptide at a dose of 10 nmol completely blocked the stimulatory effect of 0.1 nmol NPS on locomotor activity.

Neuropeptide S (NPS) was identified as the endogenous ligand of an orphan receptor now referred to as the NPS receptor (NPSR). In the frame of a structure-activity study performed on NPS Gly5, the NPSR ligand [d-Cys(tBu)5]NPS was identified. [d-Cys(tBu)5]NPS up to 100 μM did not stimulate calcium mobilization in human embryonic kidney (HEK) 293 cells stably expressing the mouse NPSR; however, in a concentration-dependent manner, the peptide inhibited the stimulatory effects elicited by 10 and 100 nM NPS (pKB, 6.62). In Schild analysis experiments [d-Cys(tBu)5]NPS (0.1–100 μM) produced a concentration-dependent and parallel rightward shift of the concentration-response curve to NPS, showing a pA2 value of 6.44. Ten micromolar [d-Cys(tBu)5]NPS did not affect signaling at seven NPSR unrelated G-protein-coupled receptors. In the mouse righting reflex (RR) recovery test, NPS given at 0.1 nmol i.c.v. reduced the percentage of animals losing the RR in response to 15 mg/kg diazepam and their sleeping time. [d-Cys(tBu)5]NPS (1–10 nmol) was inactive per se but dose-dependently antagonized the arousal-promoting action of NPS. Finally, NPSR-deficient mice were similarly sensitive to the hypnotic effects of diazepam as their wild-type littermates. However, the arousal-promoting action of 1 nmol NPS could be detected in wild-type but not in mutant mice. In conclusion, [d-Cys(tBu)5]NPS behaves both in vitro and in vivo as a pure and selective NPSR antagonist but with moderate potency. Moreover, using this tool together with receptor knockout mice studies, we demonstrated that the arousal-promoting action of NPS is because of the selective activation of the NPSR protein.

Neuropeptide S (NPS) has been identified as the endogenous ligand of a previously orphan receptor now named NPSR. Previous studies demonstrated that the N-terminal sequence Phe(2)-Arg(3)-Asn(4) of the peptide is crucial for biological activity. Here, we report on a focused structure-activity study of Arg(3) and Asn(4) that have been replaced with a series of coded and non-coded amino acids. Thirty-eight human NPS analogues were synthesized and pharmacologically tested for intracellular calcium mobilization using HEK293 cells stably expressing the mouse NPSR. The results of this study demonstrated the following NPS position 3 structure-activity features: (i) the guanidine moiety and its basic character are not crucial requirements, (ii) an aliphatic amino acid with a linear three carbon atom long side chain is sufficient to bind and fully activate NPSR, (iii) the receptor pocket allocating the position 3 side chain can accommodate slightly larger side chains at least to a certain degree [hArg, Arg(NO2) or Arg(Me)2 but not Arg(Tos)]. Position 4 seems to be more sensitive to amino acids replacement compared to position 3; in fact, all the amino acid replacements investigated produced either an important decrease of biological activity or generated inactive derivatives suggesting a pivotal role of the Asn(4) side chain for NPS bioactivity.

The spatial distribution of mast cells inside the tumor stroma has been little investigated. In this study, we have evaluated tumor mast cells distribution through the analysis of the morphological features of the spatial patterns generated by these cells, including size, shape, and architecture of the cell pattern. We have compared diffuse large B cells lymphoma (DLBCL) and systemic mastocytosis in two different anatomical localizations (lymph nodes for DLBCL and, respectively, bone marrow for mastocytosis). Results have indicated that, despite the high difference in size exhibited by the mast cells patterns in the two conditions, the spatial relationship between the mast cells forming the aggregates resulted similar, characterized by a significant tendency of the mast cells to self-organize in clusters.

The aim of the present study was to investigate the pharmacological activity of Pronetupitant, a novel compound designed to act as prodrug of the NK1 antagonist Netupitant. In receptor binding experiments Pronetupitant displayed high selectivity for the NK1 receptor. In a calcium mobilization assay performed on CHONK1 cells Pronetupitant (100 nM, 15 min preincubation) behaved as an NK1 antagonist more potent than Netupitant (pK(B) 8.72 and 7.54, respectively). In the guinea pig ileum bioassay Pronetupitant antagonized the contractile effect of SP showing a similar potency as Netupitant (pK(B)≈9). Similar results were obtained with 5 min preincubation time while at 2 min only Pronetupitant produced significant effects. In vivo in mice the intrathecal injection of 0.1 nmol SP elicited the typical scratching, biting and licking (SBL) nociceptive response. This effect of SP was dose dependently (0.1-10 mg/kg) antagonized by Pronetupitant given intravenously 2 h before the peptide. Superimposable results were obtained using Netupitant. Pharmacokinetic studies performed in rats demonstrate that Pronetupitant, after i.v. administration, is quickly (few minutes) and completely converted to Netupitant. Collectively the present results indicated that Pronetupitant acts in vitro as selective NK1 antagonist more potent than Netupitant. However based on the short half-life measured for Pronetupitant in rats, the in vivo action of Pronetupitant can be entirely interpreted as due to its conversion to Netupitant.

'Non-traditional' large neurons of the granular layer of the cerebellar cortex include all its large neuronal types, except the Golgi neuron, which is instead one of the five 'classic' types of corticocerebellar neurons. The morphological, chemical and functional characteristics of the 'non-traditional' large neurons have not been entirely ascertained. The aim of this study was to ascertain whether morphological evidence can be provided of GABA synthesis within the 'non-traditional' large neurons of the human cerebellar cortex by means of immunocytochemistry for glutamic acid decarboxylase (GAD). Fragments of postmortem cerebellar cortex of various lobules from the hemispheres and vermis were studied. Immunoreactions revealed large neurons distributed throughout the granular layer in all lobules examined. They were discriminated by analyzing the morphological features of their bodies and processes and were identified as Golgi neurons and as some 'non-traditional' types, such as the candelabrum, Lugaro and synarmotic neurons. In addition, immunoreactive large neurons, with their bodies and processes closely adjacent to microvessels, were observed throughout the layer: these perivascular neurons could represent a new type of 'non-traditional' neuron of the cerebellar cortex. This study supplies the first indication that in the human cerebellar cortex some types of 'non-traditional' large neurons are GAD-immunoreactive, in addition to those neurons already known to be GABAergic (i.e., stellate, basket, Purkinje and Golgi neurons). These morphological data further point out possible functional roles for GABA as a neurotransmitter/neuromodulator in intrinsic, associative and projective circuits of the cerebellar cortex.

1. Nociceptin/orphanin FQ (NC) and its receptor (OP4) represent a novel peptide/receptor system pharmacologically distinct from classical opioid systems. 2. Via OP4 receptor activation, NC regulates several biological functions, both at peripheral and central levels; therefore, the OP4 receptor may be viewed as a novel target for drug development. However, the pharmacology of this receptor is still in its infancy, with few molecules interacting selectively with this receptor. 3. In the present article, we review the findings of studies that have investigated the pharmacological profile of ligands selective for the OP4 receptor, these being two antagonists, the peptide [Nphe1]NC(1-13)NH2 and the non-peptide J-113397, and two agonists, the peptide [Arg14,Lys15]NC, and the non- peptide Ro 64-6198. 4. The results of these studies indicate that agents that selectively activate or block the OP4 receptor may represent new potential drugs for the treatment of human diseases.

Objective:Caregivers playa key role in the managementofpatients with cancer.However, somestudies have suggested that caregivers have even more unmet needs than the patients.Method: To better identify the needs and changes in the lifestyles of the caregivers in ourpractice and to plan a targeted support project to decrease caregiver burden, we administeredthe Caregiver’s QoL Index–Cancer (CQoLC) to 200 consecutive caregivers. This questionnaireassesses psychological well-being, the relationship with healthcare professionals,administration of finances, lifestyle disruption, and positive adaptation.Results: Our data showed that being a caregiver to a patient with metastatic diseasenegatively affected females mostly with regard to mental and emotional burden, while mencomplained more about their sexual life (42.3 vs. 33.6%), although this result was notsignificant. Some 93.5% of caregivers reported that they were pleased with their role, while83.4% were concerned about financial difficulties.Significance of results: We strongly believe that early supportive care directed not only atpatients but also to caregivers may improve the quality of life (QoL) in this population. We arecurrently developing a targeted support project to decrease caregiver burden.KEYWORDS: Cancer, Caregivers, Psychological support, Quality of lifeINTRODUCTIONWe would like to start with the following thoughtfrom John Marshall: “What we forget all too often isthe person sitting next to the patient. (. . .) Lovethat caregiver just as much as you love that patient”(Marshall, 2011). With more than 1.5 million newcancer diagnoses each yearand more than 12 millioncancer survivors (American Cancer Society, 2012), itis estimated that two of every three American famil-ieswillhaveatleastonememberdiagnosedwithcan-cer at some point.In 2012, 364.000 new cancers (excluding skin car-cinomas) were diagnosed in Italy, and there weremorethan2millionItalianswhohadapreviousdiag-nosis of cancer (AIOM–AIRTUM, 2012). In responseto these impressive data, the National Comprehen-sive Cancer Network (NCCN) Guidelines on Survi-vorship recently provided the following definition ofa cancer survivor: “An individual is considered a

Calbindin-D28k (CB) is a calcium-binding protein largely distributed in the cerebellum of various species of vertebrates. As regards the human cerebellar cortex, precise data on the distribution of CB have not yet been reported. Aim of the present work was to analyze the distribution of CB in postmortem samples of human cerebellar cortex using light microscopy immunohistochemical techniques. Immunoreactivity to CB was detected within neuronal bodies and processes distributed in all cortex layers. In the molecular layer, the immunoreactivity was observed in subpopulations of stellate and basket neurons. In the Purkinje neuron layer, the immunoreactivity was observed in practically all the Purkinje neurons. In the granular layer, the immunoreactivity was observed in subpopulations of granules, of Golgi neurons, and also of other types of large neurons (candelabrum, Lugaro neurons, etc.). Immunoreactivity to CB was also observed in axon terminals distributed throughout the cortex according to layer-specific patterns of distribution. The qualitative and quantitative patterns of distribution of CB showed no difference among the different lobes of the cerebellar cortex. This study reports that CB is expressed by different neuron types, both inhibitory (GABAergic) and excitatory (glutamatergic), involved in both intrinsic and extrinsic circuits of the human cerebellar cortex. The study provides further insights on the functional role of CB and on the neuronal types of the cerebellar cortex in which it is expressed.

A total of 32 compounds was prepared to investigate the functional role of Phe(4) in NC(1-13)-NH(2), the minimal sequence maintaining the same activity as the natural peptide nociceptin. These compounds could be divided into three series in which Phe(4) was replaced with residues that would (i) alter aromaticity or side chain length, (ii) introduce steric constraint, and (iii) modify the phenyl ring. Compounds were tested for biological activity as (a) inhibitors of the electrically stimulated contraction of the mouse vas deferens; (b) competitors of the binding of [(3)H]-NC-NH(2) to mouse brain membranes; and (c) inhibitors of forskolin-stimulated cAMP accumulation in CHO cells expressing the recombinant human OP(4) receptor. Results indicate that all compounds of the first and second series were inactive or very weak with the exception of [N(CH(3))Phe(4)]NC(1-13)-NH(2), which was only 3-fold less potent than NC(1-13)-NH(2). Compounds of the third series showed higher, equal, or lower potencies than NC(1-13)-NH(2). In particular, [(pF)Phe(4)]NC(1-13)-NH(2) (pF) and [(pNO(2))Phe(4)]NC(1-13)-NH(2) (pNO(2)) were more active than NC(1-13)-NH(2) by a factor of 5. In the mVD, these compounds showed the following order of potency: (pF) = (pNO(2)) > or = (pCN) > (pCl) > (pBr) > (pI) = (pCF(3)) = (pOCH(3)) > (pCH(3)) > (pNH(2)) = (pOH). (oF) and especially (mF) maintained high potencies but were less active than (pF). Similar orders of potency were observed in binding competition and cAMP accumulation studies. There was a strong (r(2) > or = 0.66) correlation between data observed in these assays. Biological activity data of compounds of the third series were plotted against some Hansch parameters that are currently used to quantify physicochemical features of the substituents. In the three biological assays agonist potency/affinity positively correlates with the electron withdrawal properties of the groups in the p-position of Phe(4) and inversely with their size.

The role of the amino acids contained in the sequence of HOE 140 (H-DArg(1)-Arg(2)-Pro(3)-Hyp(4)-Gly(5)-Thi(6)-Ser(7)-DTic(8)-Oic(9 )-Arg(10)-OH), a potent and selective bradykinin B(2) receptor peptide antagonist, has been investigated by the replacement of each original residue (one by one) with Ala. The resulting set of decapeptides has been tested for the B(2) antagonist activity as well as for competition with the binding of [3H]BK to plasma membranes of the human umbilical vein (hUV). Positive correlations have been established between data obtained with the bioassay and with the binding in the hUV (same species, same tissue) and also between the two bioassays, the guinea-pig ileum (GPI) and the hUV (different species, different tissue). The structure-activity study has shown that the replacement of any of the residues that constitute HOE 140 with Ala is accompanied by a decrease of potency of at least 1 log unit. The analogues can be divided into three groups, with Ala(1) and Ala(7) showing affinities lower than HOE 140 by a factor of 10, Ala(4) and Ala(10) by a factor of 100 and Ala(2), Ala(5), Ala(6), Ala(8) and Ala(9) by a factor higher than 100 (100-1000). To verify the effect of chirality, the DAla(5) and DSer(7) analogues were synthesized and it was found that the substitution with a D-residue in position 5 is not tolerated while that in position 7 is favourable. The DSer(7) derivative is the most potent analogue found in this study: it shows potency as high as that of HOE 140 in the bioassays.

Purpose: Smooth muscle reactivity is one of the factors involved in the pathogenesis of varicose veins. We investigated the myotropic effects of the 3 main vasoconstrictor agents—norepinephrine (NE), angiotensin II (Ang II), and endothelin-1 (ET-1)—in isolated human saphenous veins. Methods: Human saphenous veins were collected from 23 patients with primary chronic venous insufficiency who underwent elective varicose vein resections and who were stratified into the following 3 groups: group 1, 7 patients in clinical class 2; group 2, 9 patients in clinical classes 3 and 4; and group 3, 7 patients in clinical classes 5 and 6. Moreover, 6 patients who underwent arterial bypass grafting procedures represented the control group. The tissues were suspended in organ baths that contained Krebs solution, and their mechanical responses were measured isometrically. The cumulative concentration–response curves to Ang II, NE, and ET-1 were performed at 90-minute intervals in each tissue. Results: In the control tissues, NE, Ang II, and ET-1 induced concentration-dependent contractions with apparent affinities (pEC50, the negative logarithm to base 10 of the molar concentration of the agonist, which produces the 50% of the maximal effect) and maximal effects (maximum effect, g of contraction) that were equal to 7.06 ± 0.23, 8.53 ± 0.34, 7.63 ± 0.10, and 2.21 ± 0.33, 1.65 ± 0.31, 2.60 ± 0.77, respectively. Two main findings were evident in comparison of varicose veins with control tissues. First, the maximum effect that was evoked by all of the stimulants was reduced progressively with the increasing severity of the disease, which raised the third group to statistical significance for both NE and Ang II (P < .05). Second, a marked reduction of Ang II apparent affinity was already evident in tissues that were taken from patients in an early stage of the disease (P < .05). Conclusion: The demonstration of a significant reduction in Ang II and NE contractile activities and the important reduction of that of ET-1 in the diseased veins as compared with the control tissues extends the previous observations regarding the impairment of smooth muscle contractility in primary chronic venous insufficiency. Moreover, the dramatic reduction of Ang II affinity, which appears in an early stage of the disease, supports the hypothesis that such abnormality within the venous wall could play a role in the pathogenesis of primary varicose vein disease. (J Vasc Surg 1998;28:855-61.)

Bradykinin and related kinins act on two receptor types, named B1 and B2. Initially identified in classical bioassays, these receptors have been cloned and characterized in binding assays performed on plasma membranes of cells expressing the native or the transfected human kinin B1 or B2 receptor types. The two classification criteria recommended by Schild, namely the order of potency of agonists and the actual affinity of antagonists have been found to be applicable for receptor classification based not on data only from bioassays but also from other approaches (binding assays, molecular biology techniques). The order of potency for agonists was found with naturally occurring peptides (the kinins, their desArg9-metabolites) and with selective agonists (e.g., [Hyp3]bradykinin, [Aib7]bradykinin): the findings obtained with agonists could be validated with various antagonists. Critical evaluation of the initial compounds, typified by D)-Arg-[Hyp3, D-Phe7]bradykinin, has indicated that they are short-acting, partial agonists, non-selective for the bradykinin B2 receptor because they can be metabolized to desArg9-fragments that act on the kinin B1 receptor. Use of such compounds has given rise to misunderstandings, especially with regard to new receptor types (e.g., type B3), the existence of which was not confirmed by molecular cloning. A second generation of antagonists, represented by D-Arg[Hyp3,Thi5,D-Tic7,Oic8]bradykinin (HOE 140) has been found resistant to degradation, long-acting in vivo, selective and specific for the B2 receptor and potent in all species tested. HOE 140 has been used successfully in basic pharmacology, in animal physiopathologies involving kinins and their receptors and even in clinical studies. A third generation of non-peptide B2 receptor antagonists, whose prototype is FR 173657 ((E)-3-(6-acetamido-3-pyridyl)-N-[N-2-4-dichloro-3-[(2-methyl-8-quinolin yl)oxymethyl]phenyl]-N-methylamino carbonyl-methyl]acrylamide) is now emerging and may represent substantial progress since FR 173657 is a potent orally active, selective and specific antagonist of the human and other species B2 receptors. There is also progress regarding antagonists for the B1 receptor. The initial compounds, especially Lys-[Leu8]desArg9-bradykinin remain among of the most potent, specific and selective B1 antagonists which, however, show partial agonistic effects in some B1 receptor subtypes (e.g., the mouse). Progress has been made with AcLys-[D-betaNal7, Ile8]desArg9-bradykinin (R 715) and Lys-Lys-[Hyp3, Cpg5, D-Tic7,Cpg8]desArg9-bradykinin (B 9958) which are pure B1 antagonists in humans and rabbits; both peptides have shown resistance to degradation by peptidases and have little if any, residual agonistic activity on mouse and rat B1 receptors. No non-peptide antagonists are yet available for the B1 receptor.

The pharmacological activity of the novel neuropeptide S (NPS) receptor (NPSR) ligands QA1 and PI1 was investigated. In vitro QA1 and PI1 were tested in calcium mobilization studies performed in HEK293 cells expressing the recombinant mouse (HEK293mNPSR) and human (HEK293hNPSRIle107 and HEK293hNPSRAsn107) NPSR receptors. In vivo the compounds were studied in mouse righting reflex (RR) and locomotor activity (LA) tests. NPS caused a concentration dependent mobilization of intracellular calcium in the three cell lines with high potency (pEC50 8.73-9.14). In inhibition response curve and Schild protocol experiments the effects of NPS were antagonized by QA1 and PI1. QA1 displayed high potency (pKB 9.60-9.82) behaving as a insurmountable antagonist. However in coinjection experiments QA1 produced a rightward swift of the concentration response curve to NPS without modifying its maximal effects; this suggests that QA1 is actually a slow dissociating competitive antagonist. PI1 displayed a competitive type of antagonism and lower values of potencies (pA2 7.74-8.45). In vivo in mice NPS (0.1 nmol, i.c.v.) elicited arousal promoting action in the RR assay and stimulant effects in the LA test. QA1 (30 mgkg(-1)) was able to partially counteract the arousal promoting NPS effects, while PI1 was inactive in the RR test. In the LA test QA1 and PI1 only poorly blocked the NPS stimulant action. The present data demonstrated that QA1 and PI1 act as potent NPSR antagonists in vitro, however their usefulness for in vivo investigations in mice seems limited probably by pharmacokinetic reasons.

Species-related specific differences in the pharmacological profile of vasopressin V1a receptors have been reported. Thus, the aim of the present study was to identify a vascular preparation of human origin expressing V1a receptors. Vasopressin was found to contract human gastric artery strips without endothelium with high affinity (pEC50 8.9). The maximal effect induced by vasopressin was inversely related to the diameter of the vessel. Oxytocin was found to contract the human gastric artery strips with low potency (pEC50 7.2). Contraction induced by vasopressin was competitively antagonized by the non peptide vasopressin receptor antagonists SR 49059 (pA2 9.2), OPC 21268 (pA2 6.2) and OPC 31260 (pA2 7.1). The order of potency of agonists (vasopressin > > oxytocin) and of antagonists (SR 49059 > > OPC 31260 > OPC 21268) indicate the contraction induced by vasopressin in the isolated human gastric artery is mediated by the V1a receptor type. The present data are similar to those obtained in different preparations expressing the native human V1a receptor as well as to those obtained in cell transfected with this receptor. The human gastric artery is a monoreceptor system of great utility for studying the effects of new drugs interacting with the human V1a receptor.

The novel NK(1) receptor ligand Netupitant has been characterized in vitro and in vivo. In calcium mobilization studies CHO cells expressing the human NK receptors responded to a panel of agonists with the expected order of potency. In CHO NK(1) cells Netupitant concentration-dependently antagonized the stimulatory effects of substance P (SP) showing insurmountable antagonism (pK(B) 8.87). In cells expressing NK(2) or NK(3) receptors Netupitant was inactive. In the guinea pig ileum Netupitant concentration-dependently depressed the maximal response to SP (pK(B) 7.85) and, in functional washout experiments, displayed persistent (up to 5h) antagonist effects. In mice the intrathecal injection of SP elicited the typical scratching, biting and licking response that was dose-dependently inhibited by Netupitant given intraperitoneally in the 1-10mg/kg dose range. In gerbils, foot tapping behavior evoked by the intracerebroventricular injection of a NK(1) agonist was dose-dependently counteracted by Netupitant given intraperitoneally (ID(50) 1.5mg/kg) or orally (ID(50) 0.5mg/kg). In time course experiments in gerbils Netupitant displayed long lasting effects. In all the assays Aprepitant elicited similar effects as Netupitant. These results suggest that Netupitant behaves as a brain penetrant, orally active, potent and selective NK(1) antagonist. Thus this molecule can be useful for investigating the NK(1) receptor role in the control of central and peripheral functions. Netupitant has clinical potential in conditions such as chemotherapy induced nausea and vomiting, in which the blockade of NK(1) receptors has been demonstrated valuable for patients.

Neuropeptide S (NPS) regulates various biological functions by selectively activating the NPS receptor (NPSR). Recently, the NPSR ligand [(t)Bu-D-Gly(5)]NPS was generated and in vitro characterized as a pure antagonist at the mouse NPSR. In the present study the pharmacological profile of [(t)Bu-D-Gly(5)]NPS has been investigated. [(t)Bu-D-Gly(5)]NPS activity was evaluated in vitro in the calcium mobilization assay at the rat NPSR and in vivo in the locomotor activity and righting reflex tests in mice and in the elevated plus maze and defensive burying assays in rats. In vitro, [(t)Bu-D-Gly(5)]NPS was inactive per se while it inhibited the calcium mobilization induced by 30 nM NPS (pK(B) 7.42). In Schild analysis experiments [(t)Bu-D-Gly(5)]NPS (0.1-10 μM) produced a concentration-dependent rightward shift of the concentration-response curve to NPS, showing a pA(2) value of 7.17. In mouse locomotor activity experiments, supraspinal injection of [(t)Bu-D-Gly(5)]NPS (1-10 nmol) dose dependently counteracted NPS (0.1 nmol) stimulant effects. In the mouse righting reflex assay [(t)Bu-D-Gly(5)]NPS (0.1-10 nmol) fully prevented the arousal-promoting action of the natural peptide (0.1 nmol). Finally, [(t)Bu-D-Gly(5)]NPS (3-30 nmol) was able to completely block NPS (1 nmol) anxiolytic-like actions in rat elevated plus maze and defensive burying assays. Collectively, the present results demonstrated that [(t)Bu-D-Gly(5)]NPS behaves both in vitro and in vivo as a pure and potent NPSR antagonist. This compound represents a novel and useful tool for investigating the pharmacology and neurobiology of the NPS/NPSR system.

Aims and background The aim of the study was to evaluate the attitude at our institution in using chemotherapy at the end of life in oncology patients. We compared our habits with other clinical patterns in medical oncology, calculating the temporal interval between the last chemotherapy administration and death of the patient. Patients and methods We selected and analyzed 102 patients who received chemotherapy for metastatic or advanced solid tumors (breast, colon, gastric, pancreatic and lung cancers) and who died either in or out of a hospital or hospice from June 2007 to the end of 2009. Results We compared 51 patients enrolled in clinical trials with 51 patients not enrolled in clinical trials. Patients of both groups died with advanced cancer between June 2007 and 2009. The following solid tumor types were represented: 48% colorectal cancer, 22% breast cancer, 30% other solid tumors (pancreatic, lung and gastric cancer). The median age at death was 62 years (range, 39 to 84), the male/female ratio was 52:50, and 69% of the patients were married. Most patients, 54%, received 2–3 lines of chemotherapy, 25% received more than 3 lines, and the remaining 21% one line only of chemotherapy. Of the 102 patients identified, 16 (16%) received chemotherapy in the last month of life, and 6 (6%) of these in the last 2 weeks. We speculated that the presence of palliative care services in the territory of residence of patients could influence the time interval between the last chemotherapy and death. We found that 52 patients (51%) lived in areas where palliative care services were not available, 27 (52%) of them received chemotherapy in the last 3 months, 8 (15%) in the last month, and 5(10%) within the last 2 weeks of life. In contrast, of the 49 patients living in the territory served by palliative care units or a hospice, none received chemotherapy during the last 2 weeks of life and 37% received it during the last 3 months of life (P = 0.003). Conclusions Among selected patients who died for advanced cancer in our Operative Unit from 2007 to 2009, 50% received chemotherapy in the last 3 months of life. The availability of palliative care services in the territory of residence of patients can influence the interval between the last chemotherapy administration and death. Free full text available at www.tumorionline.it

Nociceptin/orphanin FQ (N/OFQ) controls several biological functions via selective activation of the N/OFQ peptide receptor (NOP). [(pF)Phe(4) Aib(7) Arg(14) Lys(15) ]N/OFQ-NH(2) (UFP-112) is an NOP receptor ligand designed using a combination of several chemical modifications in the same peptide sequence that increase NOP receptor affinity/potency and/or reduce susceptibility to enzymatic degradation. In the present review article, we summarize data from the literature and present original findings on the in vitro and in vivo pharmacological features of UFP-112. Moreover, important biological actions and possible therapeutic indications of NOP receptor agonists are discussed based on the results obtained with UFP-112 and compared with other peptide and nonpeptide NOP receptor ligands.

Neuropeptide S (NPS) regulates various biological functions by selectively activating the NPS receptor (NPSR). Previous studies demonstrated that the non-peptide molecule SHA 68 acts as a selective NPSR antagonist. In the present study the pharmacological profile of SHA 68 has been further investigated in vitro and in vivo. In cells expressing the mouse NPSR SHA 68 was inactive per se up to 10microM while it antagonized NPS-stimulated calcium mobilization in a competitive manner showing a pA(2) value of 8.06. In the 10-50mg/kg range of doses, SHA 68 counteracted the stimulant effects elicited by NPS, but not those of caffeine, in mouse locomotor activity experiments. In the mouse righting reflex assay SHA 68 fully prevented the arousal-promoting action of the peptide. The anxiolytic-like effects of NPS were slightly reduced by SHA 68 in the mouse open field, fully prevented in the rat elevated plus maze and partially antagonized in the rat defensive burying paradigm. Finally, SHA 68 was found poorly active in antagonizing the NPS inhibitory effect on palatable food intake in rats. In all assays SHA 68 did not produce any effect per se. In conclusion, the present study demonstrated that SHA 68 behaves as a selective NPSR antagonist that can be used to characterize the in vivo actions of NPS. However the usefulness of this research tool is limited by its poor pharmacokinetic properties.

Neuropeptide S (NPS) is the last neuropeptide identified via reverse pharmacology techniques. NPS selectively binds and activates a previous orphan GPCR, now named NPSR, producing intracellular calcium mobilization and increases in cAMP levels. Biological functions modulated by the NPS/NPSR system include anxiety, arousal, locomotion, food intake, memory, and drug addiction. The primary sequence of NPS (in humans SFRNGVGTGMKKTSFQRAKS) is highly conserved among vertebrates especially at the N-terminus. Ala- and D-scan studies demonstrated that this part of the molecule is crucial for biological activity. Focused structure-activity studies performed on Phe(2), Arg(3), and Asn(4) confirmed this indication and revealed the chemical requirements of these positions for NPSR binding and activation. The sequence Gly(5)-Val(6)-Gly(7) seems to be important for shaping the bioactive conformation of the peptide. Structure-activity studies on Gly(5) enabled identification of the first generation of peptidergic NPSR pure antagonists including [D-Cys(tBu)(5)]NPS and [D-Val(5)]NPS whose antagonist properties were confirmed in vivo. Finally, the pharmacological features of substituted bicyclic piperazine molecules (e.g. SHA 68 (3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide) were recently published making available the first generation of nonpeptide NPSR antagonists. The use in future studies of NPSR antagonists will be of paramount importance for understanding which biological functions are controlled by the NPS/NPSR system and for defining the therapeutic potential of selective NPSR ligands.

Although autoradiographic, reverse transcription-polymerase chain reaction and immunohistochemical studies have demonstrated receptors for vasoactive intestinal polypeptide (VIP) in the cerebellum of various species, immunohistochemistry has never shown immunoreactivity for VIP within cerebellar neuronal bodies and processes. The present study aimed to ascertain whether VIP immunoreactivity really does exist in the human cerebellum by making a systematic analysis of samples removed post-mortem from all of the cerebellar lobes. The study was carried out using light microscopy immunohistochemical techniques based on a set of four different antibodies (three polyclonal and one monoclonal) against VIP, carefully selected on the basis of control tests performed on human colon. All of the antibodies used showed VIP-immunoreactive neuronal bodies and processes distributed in the cerebellar cortex and subjacent white matter of all of the cerebellum lobes, having similar qualitative patterns of distribution. Immunoreactive neurons included subpopulations of the main neuron types of the cortex. Statistical analysis of the quantitative data on the VIP immunoreactivity revealed by the different antibodies in the different cerebellar lobes did not demonstrate any significant differences. In conclusion, using four different anti-VIP antibodies, the first evidence of VIP immunoreactivity is herein supplied in the human post-mortem cerebellum, with similar qualitative/quantitative patterns of distribution among the different cerebellum lobes. Owing to the function performed by VIP as a neurotransmitter/neuromodulator, it is a candidate for a role in intrinsic and extrinsic (projective) circuits of the cerebellum, in agreement with previous demonstrations of receptors for VIP in the cerebellar cortex and nuclei. As VIP signalling pathways are implicated in the regulation of cognitive and psychic functions, cerebral blood flow and metabolism, processes of histomorphogenesis, differentiation and outgrowth of nervous tissues, the results of this study could be applied to clinical neurology and psychiatry, opening new perspectives for the interpretation of neurodevelopment disorders and development of new therapeutic strategies in cerebellar diseases.

Twelve derivatives of the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) antagonist 1-benzyl-N-{3-[spiroisobenzofuran-1(3H),4′-piperidin-1-yl]propyl} pyrrolidine-2-carboxamide (Comp 24) were synthesized and tested in binding experiments performed on CHOhNOP cell membranes. Among them, a novel interesting NOP receptor antagonist (compound 35) was identified by blending chemical moieties taken from different NOP receptor ligands. In vitro in various assays, Compound 35 consistently behaved as a pure, highly potent (pA2 in the range 8.0–9.9), competitive and NOP selective antagonist. However compound 35 was found inactive when challenged against N/OFQ in vivo in the mouse tail withdrawal assay. Thus, the usefulness of the novel NOP ligand compound 35 is limited to in vitro investigations.

Neuropeptide S (NPS) regulates various biological functions by activating the NPS receptor (NPSR). Previous studies demonstrated that the substitution of Gly(5) with d-amino acids generates NPSR antagonists. Eleven [d-Xaa(5)]NPS derivatives were synthesized and pharmacologically tested measuring [Ca(2+)](i) in HEK293(mNPSR) cells. The results confirmed that the [d-Xaa(5)] substitution promotes antagonist activity with potency inversely related to the side chain size and allowed identification of the novel potent NPSR peptide antagonist [(t)Bu-d-Gly(5)]NPS.

Neuropeptide S (NPS) has been recently identified as the endogenous ligand of a previously orphan G-protein-coupled receptor now named NPSR. Both NPS and its receptor are expressed in the brain, where they modulate different functions. In particular, it has been demonstrated that intracerebroventricular (i.c.v.) injection of NPS in rodents increases wakefulness and promotes anxiolytic-like effects. In the present study we used the defensive burying (DB) test in rats to further investigate the action of human NPS (0.1-10 nmol, i.c.v.) on anxiety-related behaviors. Diazepam (1.5mg/kg, i.p.) and caffeine (20mg/kg, i.p.) were used in parallel experiments as standard anxiolytic and anxiogenic drugs, respectively. None of the tested drugs produced statistical differences in the latency to contact the probe, burying behavior latency, number of shocks received or immobility/freezing duration. Caffeine increased cumulative burying behavior and the buried bedding height in a statistically significant manner thus promoting anxiogenic like effects. Opposite results were obtained with diazepam that significantly reduced these behavioral parameters. The anxiolytic-like action of diazepam was mimicked by NPS that reduced cumulative burying behavior in a dose dependent manner. Collectively, robust anxiolytic-like effects were recorded in response to NPS in the DB test. These results are of particular interest since the outcome of this assay is marginally influenced by drug effects on locomotor activity. In conclusion, we provide further evidence that NPS evokes genuine anxiolytic-like effects in the rat; therefore NPSR selective agonists are worthy of development as innovative drugs for the treatment of anxiety disorders.

Neuropeptide S (NPS) has been identified as the endogenous ligand of a previously orphan receptor now named NPSR. Previous studies demonstrated that the N-terminal sequence Phe (2)-Arg(3)-Asn(4) of the peptide is crucial for biological activity. Here we report on a focused structure-activity study of Phe(2) which has been replaced with a series of coded and noncoded amino acids. Thirty-one human NPS analogues were synthesized and pharmacologically tested for intracellular calcium mobilization by using HEK293 cells stably expressing the mouse NPSR. The results of this study demonstrated the following NPS position 2 structure-activity features: (i) lipophilicity but not aromaticity is crucial, (ii) both the size of the chemical moiety and its distance from the peptide backbone are important for biological activity, and (iii) this position plays a role in both receptor binding and activation, since [4,4'-biphenyl-Ala(2)]hNPS behaved as a partial agonist.

The compound SB-612111 [(-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol] was recently identified as a selective antagonist for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP). In the present study, the in vitro pharmacological profile of SB-612111 at human recombinant NOP receptors expressed in Chinese hamster ovary (CHO) cells [receptor binding, guanosine 5'-O-(3-[(35)S]thio)triphosphate (GTPgamma[(35)S]) binding, and cAMP level experiments] as well as at native NOP receptors expressed in peripheral (mouse and rat vas deferens, guinea pig ileum) and central (mouse cerebral cortex synaptosomes releasing [(3)H]5-HT) preparations was evaluated and compared with that of the standard nonpeptide antagonist (+/-)J-113397 [(+/-)-trans-1-[1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one]. SB-612111 produced a concentration-dependent displacement of [(3)H]N/OFQ binding to CHO(hNOP) cell membranes, showing higher affinity and NOP selectivity over classical opioid receptors than (+/-)J-113397. SB-612111 and (+/-)J-113397 competitively antagonized the effects of N/OFQ on GTPgamma[(35)S] binding in CHO(hNOP) cell membranes (pK(B), 9.70 and 8.71, respectively) and on cAMP accumulation in CHO(hNOP) cells (pK(B), 8.63 and 7.95, respectively), being per se inactive. In isolated peripheral tissues of mice, rats, and guinea pigs and in mouse cerebral cortex synaptosomes preloaded with [(3)H]5-HT, SB-612111 competitively antagonized the inhibitory effects of N/OFQ, with pA(2) values in the range of 8.20 to 8.50. In parallel experiments, (+/-)J-113397 was found to be 2- to 9-fold less potent than SB-612111. In the electrically stimulated tissues, 1 microM SB-612111 did not modify the effects of classical opioid receptor agonists. In conclusion, the results of the present study demonstrated that SB-612111 is among the most potent and NOP-selective nonpeptide antagonists identified to date.

Neuropeptide S (NPS) has been recently recognized as the endogenous ligand for the previous orphan G-protein-coupled receptor GPR154, now referred to as the NPS receptor (NPSR). The NPS-NPSR receptor system regulates important biological functions such as sleeping/wakening, locomotion, anxiety, and food intake. To collect information on the mechanisms of interaction between NPS and its receptor, a classical structure-activity relationship study was performed. Human (h) NPS derivatives obtained by Ala and d-scan and N- and C-terminal truncation were assessed for their ability to stimulate calcium release in HEK293 cells expressing the human recombinant NPSR. The results of this study indicate that (i) the effect of hNPS is mimicked by the fragment hNPS-(1-10); (ii) Phe(2), Arg(3), and Asn(4) are crucial for biological activity; (iii) the sequence Thr(8)-Gly(9)-Met(10) is important for receptor activation, although with non-stringent chemical requirements; and (iv) the sequence Val(6)-Gly(7) acts as a hinge region between the two above-mentioned domains. However, the stimulatory effect of hNPS given intracerebroventricularly on mouse locomotor activity was not fully mimicked by hNPS-(1-10), suggesting that the C-terminal region of the peptide maintains importance for in vivo activity. In conclusion, this study identified the amino acid residues of this peptide most important for receptor activation.

Nociceptin/orphanin FQ modulates various biological functions at central and peripheral levels by selectively activating a G‐protein coupled receptor named N/OFQ peptide (NOP) receptor. For extending our knowledge on the biological roles of the N/OFQ ‐ NOP receptor system the identification of selective NOP ligands, especially antagonists, is mandatory. [Nphe(1), Arg(14), Lys(15)] N/OFQ‐NH(2) (UFP‐101) is a novel NOP ligand that was designed by combining, in the same molecule, the [Nphe(1)] chemical modification which eliminates efficacy and the [Arg(14), Lys(15)] substitution which increases ligand potency and duration of action in vivo. In the present article, we summarize the pharmacological features of UFP‐101 as determined in a series of in vitro and in vivo assays. Moreover, some biological actions and possible therapeutic indications of NOP ligands are discussed on the basis of results obtained with UFP‐101. Data obtained with this compound were compared with those generated using other NOP antagonists, especially J‐113397 and [Nphe(1)]N/OFQ(1‐13)‐NH(2), receptor or peptide knockout mice and other pharmacological tools useful for blocking N/OFQ – NOP receptor signaling. The analysis of the available data demonstrates that UFP‐101 is a useful pharmacological tool for the investigation of the central and peripheral biological functions regulated by the N/OFQ – NOP receptor system and for defining the therapeutic potential of NOP receptor ligands

A novel ligand for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), [(pF)Phe(4),Arg(14),Lys(15)]N/OFQ-NH(2) (UFP-102), has been generated by combining in the N/OFQ-NH(2) sequence two chemical modifications, [Arg(14),Lys(15)] and [(pF)Phe(4)], that have been previously demonstrated to increase potency. In vitro, UFP-102 bound with high affinity to the human NOP receptor, showed at least 200-fold selectivity over classical opioid receptors, and mimicked N/OFQ effects in CHO(hNOP) cells, isolated tissues from various species, and mouse cortical synaptosomes releasing 5-hydroxytryptamine. UFP-102 showed similar maximal effects but higher potency (2- to 48-fold) relative to N/OFQ. The effects of UFP-102 were sensitive to NOP-selective antagonists J-113397 [(+/-)-trans-1-[1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one] (pA(2) = 7.75-8.12) and UFP-101 ([Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2))(pA(2) = 6.91-7.33) but not to naloxone, and no longer observed in tissues taken from NOP receptor knockout mice (NOP(-/-)). In vivo, UFP-102 (0.01-0.3 nmol i.c.v.) mimicked the pronociceptive action of N/OFQ (0.1-10 nmol i.c.v.) in the mouse tail withdrawal assay, displaying higher potency and longer lasting effects. The action of UFP-102 was not apparent in NOP(-/-) mice. Similar results were obtained measuring locomotor activity in mice. In conscious rats, UFP-102 (0.05 nmol i.c.v.) produced a marked and sustained decrease in heart rate, mean arterial pressure, and urinary sodium excretion and a profound increase in urine flow rate. These effects were comparable with those evoked by N/OFQ at 5 nmol. Collectively, these findings demonstrate that UFP-102 behaves as a highly potent and selective NOP receptor agonist that produces long-lasting effects in vivo.

Aims and background Recent preclinical data have suggested that lonidamine may potentiate the activity of mitomycin C in human colon cancer cell lines LoVo and HT29. Study design A phase II study was carried out in 14 patients with advanced colorectal cancer pretreated with fluorouracil and folinic acid. Treatment consisted of lonidamine, 600 mg po, followed after 2 h by mitomycin, 20 mg/m2 by iv bolus, followed by lonidamine, 150 mg tid for 5 days; the cycle was repeated every 6 weeks. Results No objective response was seen. Three patients had stable disease; the median survival for the whole group was 4 months. Although hematologic toxicity was negligible, lonidamine-related side effects were moderate to severe in most patients and mainly represented by myalgia and gastric pain. Discussion Despite a sound preclinical rationale, this schedule of lonidamine and mitomycin C was ineffective and toxic in patients with advanced colorectal cancer. More experimental data about lonidamine are needed in order to design more effective regimens based on the combination of this interesting drug with other anticancer agents.

In this study, we describe the in vitro and in vivo activities of a series of cyclic peptide analogues of the selective kinin B2 receptor antagonist MEN11270 on Chinese hamster ovary cells expressing the human B2 receptor (hB2R), the human isolated umbilical vein (hUV), the isolated guinea pig ileum (gpI), and bradykinin (BK) induced bronchoconstriction (BC) and hypotension in anaesthetized guinea pigs. Substitutions in the backbone of MEN11270 (H-DArg-Arg-Pro-Hyp-Gly-Thi-c(Dab-DTic-Oic-Arg)c(7γ-10α)) aimed to increase the potency in inhibiting bronchospasm versus hypotension following the topical (intratracheal (i.t.)) or systemic (intravenous (i.v.)) application of these antagonists. A series of analogues were left unprotected from N-terminal cleavage by aminopeptidases (MEN12739, MEN13052, MEN13346, and MEN13371): these compounds maintained sizeable affinities for the hB2R (pKi = 9.4, 9.6, 9.7, and 8.6, respectively) and antagonist activities toward BK in the hUV (pA2 = 7.9, 8.3, 8.2, and 7.5) and gpI assays (pKB = 7.4, 7.8, 7.9, and 7.9), but the inhibition of BK-induced BC and hypotension in vivo was negligible following either i.v. or i.t. administration. Two analogues (MEN12388 and MEN13405) could be potential substrates of angiotensin-converting enzyme: these have good activity in the hB2R (pKi = 9.5 and 8.9, respectively), hUV (pA2 = 8.2 for MEN12388), and gpI assays (pKB = 8.4 and 8.0) but an in vivo activity 10- to 30-fold lower than the parent compound MEN11270 (pKi = 9.4, pA2 = 8.1, pKB = 8.3) when given by either the i.v. or the i.t. route. Other analogues were functionalized with a quaternary ammonium Lys derivative (MEN13031, MEN12374, and the previously mentioned MEN13052) or with an ethyl group on Arg (MEN13655 and the previously mentioned MEN13346 and MEN13405) in order to hinder or facilitate local absorption. MEN13346 and MEN13031 (pKi = 9.7and 9.5, pA2 = 8.2 and 7.9, pKB = 7.9 and 8.5, respectively) were 10- to 30-fold less active in vivo than MEN11270, without improving the discrimination between BK-induced BC and hypotension after either systemic or topical administration. It is concluded that the decreased in vivo activities of cyclic analogues of MEN11270 on BK-induced BC and hypotension following either their intratracheal or their intravenous routes of administration might be due in large part to metabolic degradation.Key words: bradykinin, asthma, blood pressure, guinea pig, metabolism.

Peptide and nonpeptide compounds have been shown to interact specifically with B2 receptors of three different species, namely human, rabbit, and pig. Peptide agonists and nonpeptide antagonists show marked differences in potencies and suggest the existence of B2 receptor subtypes. This conclusion is based on data obtained with the modified agonist peptide LF 150943 whose potency (pEC50 9.4) is at least 100-fold higher in rabbit than in humans (7.4) and pig (6.7). The same conclusion can be drawn from data obtained with antagonists that are more potent in humans (LF 160687, pA2 9.2) than in rabbit (8.7) and pig (8.2) or with antagonists (S 1567) that show the opposite potency order, being much weaker in humans (pA2 6.9) than in rabbit (7.6) and pig (9.4). Two other compounds (FR 173657 and FR 172357) show similar pharmacological spectra as S 1567 and differ from LF 160687.Key words: bradykinin, B2 receptor ligands, bioassay, isolated vessels.

We have studied the effects of naloxone benzoylhydrazone (NalBzoH) at recombinant human OP4 receptors expressed in Chinese hamster ovary (CHO) cells (CHOhOP4) and native OP4 sites in isolated tissues from various species. In CHOhOP4 membranes, nociceptin (NC) and NalBzoH displaced [125I]Tyr14-NC with pKi values of 10.1 and 7.3. In the presence of 100 µM GDP, NC stimulated GTPγ35S binding (pEC50 = 8.5). NalBzoH was ineffective but antagonized the effects of NC (pA2 = 6.9). At 5 µM GDP, there was an increase in potency (pEC50 = 9.3) and efficacy (4.3-fold) of NC. NalBzOH was a partial agonist (pEC50 = 7.0, Emax = 13% relative to NC). In CHOhOP4 cells, NC and NalBzoH inhibited cAMP formation with pEC50 and Emax values of 9.8 and 100% and 6.0 and 44%, respectively. In the rat vas deferens, NalBzoH (10 µM) did not modify electrically induced twitches but competitively antagonized the inhibitory action of NC (pA2 = 6.2). In the mouse vas deferens (mVD) and guinea pig ileum (gpI), NalBzoH inhibited twitches with pEC50 and Emax values of 7.6 and 78% and 8.5 and 77%, respectively. The effect of 3 µM NalBzoH was fully inhibited by 3 µM naloxone in mVD and 30 µM in gpI. Under these conditions, NalBzoH antagonized the actions of NC in both preparations with pA2 values of 6.3 and 6.8, respectively. Collectively, these data demonstrate that NalBzoH is a nonselective OP4 ligand with system-dependent behaviour.Key words: nociceptin/orphanin FQ, naloxone benzoylhydrazone, OP4 receptors, cAMP, GTPγS, bioassay.

Urotensin II is a cyclic undecapeptide which activates the GPR14 receptor and exerts potent vasoconstrictor effects in some species of fish and mammals. The present study intended to investigate isolated vessels from various species in an attempt to find sensitive preparations to be used in studies of the human urotensin (hU-II)/GPR14 system. Contractile responses evoked by noradrenaline (NA), angiotensin II (Ang II), endothelin 1 (ET-1) and hU-II were measured in large vessels (aorta and some large arteries and veins) of rats, guinea pigs, rabbits, pigs and humans. Relaxing effects of hU-II, bradykinin (BK) and substance P (SP) were measured in pig coronary arteries contracted with KCl 30 mM. The rat mesenteric vasculature was investigated from the arterial and venous site to establish the function of ET-1 and hU-II receptors. Results indicate that the only preparation showing high sensitivity to hU-II (pEC(50)=8.27) is the rat aorta, whose contractions in response to hU-II develop slowly and persist for hours, similar to those of ET-1 (pEC(50)=8.35). Effects of NA (pEC(50)=8.12) and Ang II (pEC(50)=7.95) develop and reverse more rapidly. Tissues treated with ET-1 and hU-II show marked desensitization, in contrast to those treated with NA. Specific antagonists for alpha(1) (prazosin, p A(2)=10.46), AT(1) (EXP 3174, p A(2)=10.20), 5HT(2) (ketanserine, p A(2)=8.61) and ET(A)-ET(B) (bosentan, p A(2)=6.88) receptors were shown to block the effects of the respective agonists, while being inactive against hU-II. In some vessels, hU-II behaved as an highly potent but scarcely effective contractile agent. It is concluded that: the hU-II/GPR14 is not a functional contractile system in vessels of several species, in contrast with NA/alpha(1), Ang II/AT(1), 5HT/5HT(2) and ET-1/ET(A)-ET(B). The rat aorta appears however to be a sensitive and reliable preparation for evaluating biological activities of hU-II and related peptides.

Phe(4) in the nociceptin (NC) sequence has been identified as the most critical residue for receptor interaction. In the present study, we investigated the pharmacological activity of a series of NC(1-13)NH(2) analogues, in which the hydrogen atom in the para position of Phe(4) was substituted with F, NO(2), CN, Cl, Br, I, CH(3), OH or NH(2). In receptor binding studies, performed using CHO cells expressing the recombinant human NC receptor (CHO(hOP4)) and in rat cerebral cortex membranes, [(pF)Phe(4)]NC(1-13)NH(2), [(pNO(2))Phe(4)]NC(1-13)NH(2), and [(pCN)Phe(4)]NC(1-13)NH(2) displayed higher affinity than NC(1-13)NH(2). The affinity of [(pCl)Phe(4)]NC(1-13)NH(2) was essentially identical to that of NC(1-13)NH(2), while the remaining compounds displayed reduced affinity. In a series of functional assays (stimulation of GTPgammaS binding in CHO(hOP4)cells and rat cerebral cortex membranes and inhibition of cAMP accumulation in CHO(hOP4) cells), the para substituted analogues behaved as full agonists (with the exception of [(pOH)Phe(4)]NC(1-13)NH(2) which acted as a partial agonist in the GTPgammaS binding assays) with the following rank order potency:[(pF)Phe(4)]NC(1-13)NH(2) and [(pNO(2))Phe(4)]NC(1-13)NH(2) were either inactive or displayed micromolar potencies in cAMP accumulation experiments performed on cells expressing classical opioid receptors. All compounds were full agonists in isolated tissues from various species (guinea pig ileum, mouse colon and mouse/rat vas deferens) with the exception of [(pOH)Phe(4)]NC(1-13)NH(2) which displayed partial agonist/weak antagonist activities. The rank order of potency was similar to that found in the other assays. The effects of all analogues were not modified by naloxone. The selective OP(4) receptor antagonist [Nphe(1)]NC(1-13)NH(2), tested in all preparations against one or both of the highly potent derivatives [(pF)Phe(4)]NC(1-13)NH(2) and [(pNO(2))Phe(4)]NC(1-13)NH(2), showed pA(2) values similar to those found against NC, the pA(2) in the GTPgammaS binding/rat cerebral cortex assay being much higher (ca. 7.5) than in the other functional assays (ca. 6). This study further supports the notion that Phe(4) of NC is the critical residue for receptor occupation and activation. Moreover, as part of this study, we have identified two novel, highly potent and selective agonists for the OP(4) receptor, [(pF)Phe(4)]NC(1-13)NH(2) and [(pNO(2))Phe(4)]NC(1-13)NH(2).

This minireview is divided into three parts: the first part refers to the characterization and classification of kinin receptors using agonists and antagonists in isolated tissues (classical pharmacology). Two kinin receptors have been considered on the basis of their distinct pharmacology, namely the B1receptor of the rabbit aorta (rank order of potency of agonists: LysdesArg9BK > desArg9BK >= LysBK>BK; apparent affinities of antagonists Lys[Leu8]desArg9BK (pIC508.4 ) > [Leu8]desArg9BK (pIC507.4)>>>HOE 140, a B2receptor antagonist, pIC502receptor of the rabbit jugular vein (potency of agonists: LysBK = BK>>> LysdesArg9BK = desArg9BK and HOE 140 (pIC509.0)>>>Lys[Leu8]desArg9BK, pIC501receptor subtypes, demonstrated by different pharmacological profiles of agonists and antagonists: human, rabbit and pig subtypes (LysdesArg9BK >> desArg9B K and Lys[Leu8]desArg9BK > [Leu8]desArg9BK) and dog, rat, mouse and hamster B1receptors (desArg9BK = LysdesArg9BK and [Leu8]desArg9BK = Lys[Leu8]desArg9BK). Affinities of agonists and antagonists in some species (man, rabbit, pig) are significantly increased (at least 10-fold) by the presence of a Lys at their Nterminus. The last part describes species-related B2receptor subtypes supported by results obtained with non-peptide receptor agonists (FR 190997) and antagonists (FR 173657). While BK acts as a full agonist in man, rabbit and pig, FR 190997 behaves as a full agonist on human, as partial agonist on rabbit, and as pure antagonist on pig B2receptors. Various hypotheses are considered to interpret these findings.

Three series of nociceptin (NC)-related peptides were synthesized and their abilities (i) to bind to the NC sites expressed in mouse forebrain membranes, (ii) to inhibit the electrically evoked contraction of the mouse vas deferens, and (iii) to inhibit forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells expressing the human recombinant NC receptor (CHO NCR ) were investigated. The compounds of the first series (a series) have an ordinary Xaa 1 -Gly 2 bond, those of the second series (b series) have a Xaa 1 Ψ(CH 2 -NH)Gly 2 pseudopeptide bond, and those of the third series (c series) have a peptoid (Nxaa 1 -Gly 2 ) structure. The affinity values measured in the binding assay and in the two functional assays with the compounds of the three series showed high levels of correlation. Thus, (I) the compounds of the a series in which Phe 1 was substituted with Tyr, Cha, or Leu acted as potent NC receptor agonists; (II) the b series compounds behaved as NC receptor antagonists in the mouse vas deferens and as full agonists in CHO NCR cells with different potencies depending on the first amino acid residue, [Phe 1 Ψ(CH 2 -NH)Gly 2 ]NC(1-17)NH 2 and [Phe 1 Ψ(CH 2 -NH)Gly 2 ]NC(1-13)NH 2 being the most potent compounds; (III) the compounds of the third series were all inactive both as agonists and as antagonists with the exception of [Nphe 1 ]NC(1-17)NH 2 and [Nphe 1 ]NC(1-13)NH 2 , which behaved as NC receptor antagonists both in the isolated tissue and in CHO NCR cells (pK B 6.1-6.4). In conclusion, this study demonstrates that chemical requirements for NC receptor agonists are different from those of antagonists. Moreover, modifications of the steric orientation of the aromatic residue Phe 1 in the NC sequence as obtained with the pseudopeptide bond between Phe 1 and Gly 2 or with the displacement of the benzyl side chain by one atom, as in Nphe 1 , lead respectively to reduction or elimination of efficacy. Indeed, in contrast to [Phe 1 Ψ-(CH2-NH)Gly 2 ]NC(1-13)NH2 which has been reported to exhibit agonist activity in several assays involving either central or recombinant NC receptors, [Nphe 1 ]NC(1-13)NH 2 antagonizes the effect of NC at human recombinant NC receptors and in the mouse tail withdrawal assay.

Nociceptin (NC), alias Orphanin FQ (OFQ) is a heptadecapeptide structurally related to opioid peptides, especially Dynorphin A, which, however, does not interact with classic opioid receptors. NC selectively activates its own receptor (OP(4)), which has been shown to be insensitive to the naturally occurring opioid peptides as well as to a large number of non-peptide opioid receptor ligands, including naloxone. Thus, the NC/OP(4) system represents a new peptide-based signaling pathway, which is pharmacologically distinct from the opioid systems. The pharmacological tools available for investigating NC actions are at present rather limited and include: 1) peptide ligands obtained from structure activity studies performed using NC(1-13)NH(2) as a template or discovered by screening peptide combinatorial libraries; 2) nonpeptide ligands that are either molecules already known to interact with classic opioid receptors or novel molecules designed and synthesized as selective ligands of the OP(4) receptor. In the present paper the functional data obtained from both in vitro and in vivo studies with each relevant OP(4) receptor ligand will be analyzed and discussed comparing the advantages and disadvantages of each molecule. We hope that the present work will aid investigators, working in the NC/OP(4) field, in the choice of the pharmacological tools suitable for their experiments.

FR 190997, a new kinin B2 receptor agonist of non-peptide nature, has been studied in three isolated vessels: the human umbilical vein (hUV), the rabbit jugular vein (rbJV), and the pig coronary artery (pCA). Bradykinin (BK) contracts the hUV and rbJV through smooth muscle B2 receptors, while it relaxes the pCA through endothelial receptors of the B2 type. Contractions of the hUV and rbJV in response to FR 190997 show slow onset and are not reproducible compared to the rapid and reproducible effect of BK. They reach only 70% and 30% of the BK-induced maximal contractions in the hUV and rbJV, respectively. The effects of FR 190997 are antagonised by HOE 140 and this antagonist shows similar pK(B) values against BK and FR 190997, indicating that the non-peptide agent interacts with the kinin B2 receptor. FR 190997 is inactive as relaxant of the pCA; in this tissue, it acts as a pure and competitive antagonist, with a pK(B) value of 7.6, while HOE 140 acts as an insurmountable antagonist (pK(B) 9.3). When tested as an antagonist, FR 190997 inhibits also the contractile effects of BK in the hUV (pK(B) 7.8) and in the rbJV (pK(B) 7.6). FR 190997 is selective for the B2 receptor since it does not interact with the B1, and is specific since it does not affect the contraction evoked by 5-hydroxytryptamine, endothelin-1, and noradrenaline in the hUV, or the relaxation induced by substance P in the pCA. FR 190997 shows therefore different pharmacological profiles in various preparations, acting as a partial agonist in the hUV and especially in the rbJV and as a pure antagonist in the pCA. This new compound could be of interest in understanding how non-peptide agonists may activate receptors for peptides.