Pharmacological hypothermia induced neurovascular protection after severe stroke of transient middle cerebral artery occlusion in mice.

Therapeutic hypothermia is a potential protective strategy after stroke. The present study evaluated the neurovascular protective potential of pharmacological hypothermia induced by the neurotensin receptor 1 agonist HPI-201 after severe ischemic stroke. Adult C57BL/6 mice were subjected to filament insertion-induced occlusion of the middle cerebral artery (60 min MCAO). HPI-201 was i.p. injected 120 min after the onset of MCAO to initiate and maintain the body temperature at 32-33°C for 6 hrs. The infarct volume, cell death, integrity of the blood brain barrier (BBB) and neurovascular unit (NVU), inflammation, and functional outcomes were evaluated. The hypothermic treatment significantly suppressed the infarct volume and neuronal cell death, accompanied with reduced caspase-3 activation and BAX expression while Bcl-2 increased in the peri-infarct region. The cellular integrity of the BBB and NVU was significantly improved and brain edema was attenuated in HPI-201-treated mice compared to stroke controls. The hypothermic treatment decreased the expression of inflammatory factors including tumor necrosis factor-α (TNF-α), MMP-9, interleukin-1ß (IL-1ß), the M1 microglia markers IL-12 and inducible nitric oxide synthase (iNOS), while increased the M2 marker arginase-1 (Arg-1). Stroke mice received the hypothermic treatment showed lower neurological severity score (NSS), performed significantly better in functional tests, the mortality rate in the hypothermic group was noticeably lower compared with stroke controls. Taken together, HPI-201 induced pharmacological hypothermia is protective for different neurovascular cells after a severely injured brain, mediated by multiple mechanisms.

Evidence of 68Ga-DOTA-NT-20.3 Uptake in Pancreatic Adenocarcinoma AsPC-1 Cell Line - in vitro Study.

BACKGROUND: Neurotensin receptors are overexpressed in several cancer types including pancreatic ductal adenocarcinoma. Three NTR subtypes have been cloned: NTR-1, NTR-2 and NTR-3. The most expressed NTR-1 is not present in normal pancreatic tissue and has a low expression in chronic pancreatitis. OBJECTIVE: Objective of this study was to test in vitro affinity of the new 68Ga labelled neurotensin analogue DOTA-NT-20.3 (fragment 6-13, Ac-Lys(DOTA)-Pro-Arg(N-CH3)-Arg-Pro-Tyr-Tle-Leu) on the human pancreatic ductal adenocarcinoma cell line AsPC-1. METHOD: For the preparation of 68Ga-DOTA-NT-20.3, 68GaCl3 solution (eluted from 68Ge/68Ga generator) and 50 µg of precursor (Iason, Graz, Austria) water dissolved were used in an automatic synthesis module. The labeled compound was added to cell culture flask and incubated at 37°C. At various time points after tracer addition up to 80min, cells were recovered, rinsed and counted for radioactivity. Results were expressed as percent binding normalized to 200000 cells and affinity parameters were calculated. RESULTS: Labeling yield was ≥98 %. The molar ratio between labelled and total peptide was about 1/400. AsPC-1 cell line showed rapid uptake of the tracer including surface and internalized binding, tending to a plateau phase 80 min after tracer addition (11%/200.000 cells). The Kd (7.335 pmol) and Bmax (90.52 kBq) value indicated high tracer affinity for AsPC-1cell line especially if compared with the literature data regarding other malignancies (e.g. colonic cancer cell line). Binding sites were 1.09x106 sites per cell. CONCLUSION: New tracer 68Ga-DOTA-NT-20.3 can be a suitable candidate for the clinical use in patients with pancreatic ductal adenocarcinoma.

Structural Optimization and Characterization of Potent Analgesic Macrocyclic Analogues of Neurotensin (8-13).

The neurotensin receptors are attractive targets for the development of new analgesic compounds. They represent potential alternatives or adjuvants to opioids. Herein, we report the structural optimization of our recently reported macrocyclic peptide analogues of NT(8-13). The macrocycle was formed via ring-closing metathesis (RCM) between an ortho allylated tyrosine residue in position 11 and the side chain of alkene-functionalized amino acid in position 8 of NT(8-13). Minute modifications led to significant binding affinity improvement ( Ki improved from 5600 to 15 nM) with greatly improved plasma stability compared to NT(8-13). This study also delineates the structural features influencing these parameters. The signaling profiles of the new macrocycles were determined on the NTS1 receptor, and the physiological effects of the two most potent and stable analogues were assessed in vivo using rodent models. Both compounds displayed strong analgesic effects.

Neurotensin and neurotensin receptors: characteristic, structure-activity relationship and pain modulation--a review.

Neurotensin (NT) is a tridecapeptide, which - since its discovery in 1973--has been demonstrated to be involved in the control of various physiological activities in both the central nervous system and in the periphery. Its biological effects are mediated by four receptor types. Exogenously administered NT exerts different behavioral effects, including antinociception. Structure-activity relationship studies performed in recent years resulted in development of several peptidomimetic receptor agonists and non-peptidic receptor antagonists that are useful tools for studies of NT mechanisms in tissue and on cellular level. This may result in design of new generation of analgesics based on neurotensin. NT antinociceptive effects are distinct from opioid analgesia. This creates opportunity of development of hybride analgesics that may simultaneously activate both opioid and NT antinociceptive pathways.

The brattleboro rat displays a natural deficit in social discrimination that is restored by clozapine and a neurotensin analog.

Cognitive deficits in schizophrenia are a major source of dysfunction for which more effective treatments are needed. The vasopressin-deficient Brattleboro (BRAT) rat has been shown to have several natural schizophrenia-like deficits, including impairments in prepulse inhibition and memory. We investigated BRAT rats and their parental strain, Long-Evans (LE) rats, in a social discrimination paradigm, which is an ethologically relevant animal test of cognitive deficits of schizophrenia based upon the natural preference of animals to investigate conspecifics. We also investigated the effects of the atypical antipsychotic, clozapine, and the putative antipsychotic, PD149163, a brain-penetrating neurotensin-1 agonist, on social discrimination in these rats. Adult rats were administered saline or one of the three doses of clozapine (0.1, 1.0, or 10 mg/kg) or PD149163 (0.1, 0.3, or 1.0 mg/kg), subcutaneously. Following drug administration, adult rats were exposed to a juvenile rat for a 4-min learning period. Animals were then housed individually for 30 min and then simultaneously exposed to the juvenile presented previously and a new juvenile for 4 min. Saline-treated LE rats, but not BRAT rats, exhibited intact social discrimination as evidenced by greater time spent exploring the new juvenile. The highest dose of clozapine and the two highest doses of PD149163 restored social discrimination in BRAT rats. These results provide further support for the utility of the BRAT rat as a genetic animal model relevant to schizophrenia and drug discovery. The potential of neurotensin agonists as putative treatments for cognitive deficits of schizophrenia was also supported.

Involvement of the neurotensin receptor 1 in the behavioral effects of two neurotensin agonists, NT-2 and NT69L: lack of hypothermic, antinociceptive and antipsychotic actions in receptor knockout mice.

Neurotensin (NT) is a neuropeptide implicated in the pathophysiology of schizophrenia and in mediating the efficacy of antipsychotic drugs. NT is also involved in the regulation of body temperature and pain sensitivity. Using neurotensin receptor 1 (NTR1) knockout (KO) and wild-type (WT) mice, these studies evaluated the involvement of NTR1 in the behavioral responses produced by peripheral administration of NT agonists (NT-2 and NT69L). Animals were characterized in paradigms designed to assess hypothermia, antinociception, and antipsychotic-like effects. Under basal conditions, there were no phenotypic differences between NTR1 KO and WT mice. In WT mice, both NTR1 agonists decreased core body temperature (active doses in mg/kg, i.p., for NT-2 and NT69L, respectively: 1 and 3), increased tail withdrawal latencies (1 and 3), produced decreased spontaneous climbing (0.1, 0.3, 1 and 1, 3, 10) and reversed apomorphine-induced climbing (0.3, 1 and 1, 3). In contrast, none of the effects of either agonist were present in KO mice. These results suggest that NTR1: (1) does not play a major role in the control of basal thermoregulation, nociception or psychomotor stimulation in mice (barring possible developmental plasticity), (2) does mediate these behavioral responses to NT agonists, and (3) may play a role in the potential antipsychotic effects of these agonists.

Effect of a novel neurotensin analog, NT69L, on nicotine-induced alterations in monoamine levels in rat brain.

NT69L, is a novel neurotensin (8-13) analog that participates in the modulation of the dopaminergic pathways implicated in addiction to psychostimulants. NT69L blocks nicotine-induced hyperactivity as well as the initiation and expression of sensitization in rats. Recent evidence suggests that stimulation of mesocorticolimbic dopamine system, with influences from the other monoamine systems, e.g. norepinephrine and serotonin, is involved in nicotine's reinforcing properties. The aim of the present study was to investigate the effect of pretreatment with NT69L on nicotine-induced changes in monoamine levels in the rat brain using in vivo microdialysis. Acute or chronic (0.4 mg/kg, sc, once daily for 2 weeks) administration of nicotine elicited increases in extracellular levels of dopamine, dopamine metabolites, norepinephrine, or serotonin in medial prefrontal cortex, nucleus accumbens shell, and core of rats. Pretreatment with NT69L (1 mg/kg, intraperitoneally, ip) administered 40 min before nicotine injection significantly attenuated the acute nicotine-evoked increases in norepinephrine levels in medial prefrontal cortex, dopamine and serotonin in nucleus accumbens shell. After chronic nicotine administration, pretreatment of NT69L markedly reversed the increase in dopamine levels in the nucleus accumbens core. NT69L's attenuation of some of the biochemical effects of acute and chronic nicotine is consistent with this peptide's attenuation of nicotine-induced behavioral effects. These data further support a role for NT69L or other neurotensin receptor agonists to treat nicotine addiction.

Selectively increased trace conditioning under the neurotensin agonist PD 149163 in an aversive procedure in which SR 142948A was without intrinsic effect.

There is evidence to suggest that neurotensin (NT) may enhance cognitive function. The present study, therefore, examined the role of NT in associative learning between a conditioned stimulus (CS) and an unconditioned stimulus (UCS). This was tested in a trace procedure using conditioned suppression of drinking with a noise CS and foot shock UCS. We compared the effects of an NT agonist (PD 149163, 0.25 and 1 mg/kg) with those of an NT antagonist (SR 142948A, 0.01 and 0.1 mg/kg). Conditioning after drug treatment was followed by drug-free tests of associative learning. At 0.25 but not 1 mg/kg, PD 149163 selectively increased conditioning over the trace interval: there was no such increased conditioning in the 0s group. This increased conditioning over the trace is an effect that is reliably produced by dopamine (DA) agonists in the same procedure. However, dissimilar to the effects of DA agonists, conditioning to box context, was reduced under PD 149163. Doses of SR 142948A, selected on the basis of their effects in similar aversively motivated tests of latent inhibition, were without intrinsic effect in the present procedure. The dose-related dissociation between trace and contextual conditioning effects under PD 149163 is considered as cognitive enhancement.

Neurotensin agonists: potential in the treatment of schizophrenia.

Neurotensin (NT) is a neuropeptide that, for decades, has been implicated in the biology of schizophrenia. It is closely associated with, and is thought to modulate, dopaminergic and other neurotransmitter systems involved in the pathophysiology of various neuropsychiatric diseases, including schizophrenia. This review outlines the neurochemistry and function of the NT system and the data implicating its role in schizophrenia. The data suggest that NT receptor agonists have the potential to be used as novel therapeutic agents for the treatment of schizophrenia, with the added benefits of (i) not causing weight gain, an adverse effect that is problematic with some of the currently used atypical antipsychotic drugs; and (ii) helping patients to stop smoking, a behaviour that is highly prevalent in those with schizophrenia.

Involvement of neurotensin in cancer growth: evidence, mechanisms and development of diagnostic tools.

Focusing on the literature of the past 15 years, we evaluate the evidence that neurotensin and neurotensin receptors participate in cancer growth and we describe possible mechanisms. In addition, we review the progress achieved in the use of neurotensin analogs to image tumors in animals and humans. These exciting advances encourage us to pursue further research and stimulate us to consider novel ideas regarding the multiple inputs to cancer growth that neurotensin might influence.

Functional domains of the subtype 1 neurotensin receptor (NTS1).

The subtype 1 neurotensin receptor (NTS1) belongs to the family of G protein coupled receptors with seven transmembrane domains and mediates most of the known effects of neurotensin. In the past years, mutagenesis studies have allowed to delineate functional regions of the receptor involved in agonist and antagonist binding, G protein coupling, sodium sensitivity of agonist binding, and agonist-induced receptor internalization. These data are reviewed and discussed in the present paper.

Novel treatments of schizophrenia: targeting the neurotensin system.

Evidence implicating neural circuits that utilize the neuropeptide transmitter neurotensin (NT) in the pathophysiology of schizophrenia and in the mechanism of action of antipsychotic drugs has previously been reviewed. The majority of evidence, taken together, supports the development of NT receptor agonists as novel antipsychotic drugs. This review comprehensively describes the NT receptor subtypes, discusses the development of NT receptor agonists and the behavioral effects of currently available NT receptor agonists. The compilation of data suggests that NT receptor agonists may represent a novel class of antipsychotic drugs for the treatment of schizophrenia.

Neurotensin agonists as an alternative to antipsychotics.

Neurotensin (NT) is a 13 amino acid neuropeptide that is found in the central nervous system and in the gastrointestinal tract. In brain, this peptide is prominently associated anatomically with dopaminergic, as well as other neurotransmitter systems. Based on animal studies, already decades old, researchers have hypothesised that NT receptor agonists will have antipsychotic properties in patients. However, to date no one has obtained a non-peptide NT receptor agonist. Therefore, there has been great interest in obtaining peptide analogues of NT, that, unlike NT resist degradation by peptidases and cross the blood-brain barrier, yet have the pharmacological characteristics of native NT, for therapeutic use in the treatment of schizophrenia, as well as other neuropsychiatric diseases such as Parkinson's disease and addiction to psychostimulants. In this review, we present the rationale for development of NT receptor agonists for treatment of certain central nervous system diseases, as well as a review of those peptide agonists that are in early stages of development.

The neurotensin agonist PD149163 increases Fos expression in the prefrontal cortex of the rat.

Dopaminergic axons innervating the prefrontal cortex (PFC) target both pyramidal cells and GABAergic interneurons. Many of these dopamine (DA) axons in the rat coexpress the peptide neurotransmitter neurotensin. Previous electrophysiological data have suggested that neurotensin activates GABAergic interneurons in the PFC. Activation of D2-like DA receptors increases extracellular GABA levels in the PFC, as opposed to the striatum, where D2 receptor activation inhibits GABAergic neurons. Because activation of presynaptic D2 release-modulating autoreceptors in the PFC suppresses DA release but increases release of the cotransmitter neurotensin, D2 agonists may enhance the activity of GABAergic interneurons via release of neurotensin. In order to determine if neurotensin can activate GABAergic interneurons, we treated rats with the peptide neurotensin agonist, PD149163, and examined Fos expression in PFC neurons. Systemic administration of PD149163 increased overall Fos expression in the PFC, but not in the dorsal striatum. PD149163 induced Fos in PFC interneurons, as defined by the presence of calcium-binding proteins, and in pyramidal cells. Pretreatment with the high-affinity neurotensin antagonist, SR48692, blocked neurotensin agonist-induced Fos expression. These data suggest that neurotensin activates interneurons in the PFC of the rat.

Neurotensin agonists block the prepulse inhibition deficits produced by a 5-HT2A and an alpha1 agonist.

RATIONALE: Neurotensin (NT) agonists have been proposed as potential antipsychotics based exclusively upon their ability to inhibit dopamine-2 (D2) receptor transmission. Several other pharmacological mechanisms have been implicated in enhancing the antipsychotic profile produced by D2 inhibition alone. These include inhibition of 5-HT2A and alpha1-adrenoceptors. Recently, we reported that systemic administration of the neurotensin agonist PD149163 blocks deficits in prepulse inhibition (PPI) of the startle reflex produced by the 5-HT2A receptor agonist DOI. This suggested that NT agonists could inhibit 5-HT2A modulation of neurotransmission. OBJECTIVE: To determine if other peripherally administered NT agonists shared this effect, we examined the effects of NT69L, another NT agonist, on DOI-induced PPI deficits. In addition, to determine if NT agonists also inhibit alpha1-adrenoceptor neurotransmission, we examined the effects of PD149163 and NT69L on PPI deficits induced by the alpha1-adrenoceptor agonist, cirazoline. METHODS: In the NT69L/DOI study, rats received subcutaneous (SC) injections of NT69L (0, 0.1, 1, or 2 mg/kg) followed 30 min later by SC saline or DOI (0.5 mg/kg). In the NT agonist/cirazoline studies, animals received SC injections of either PD149163 (0, 0.01, 0.1, or 1 mg/kg) or NT69L (0, 0.01, 0.1, or 1 mg/kg) followed 30 min later by SC saline or cirazoline (0.7 mg/kg). Animals were tested in startle chambers 20 min later. RESULTS: In all three experiments the PPI disruption produced by DOI and cirazoline was blocked by the NT agonists. CONCLUSIONS: These findings provide strong evidence that NT agonists inhibit 5-HT2A and alpha1-adrenoceptor modulation of neurotransmission, pharmacological effects that, in conjunction with their known inhibition of dopamine transmission, strengthen the antipsychotic potential of NT agonists.

Reversal of sensorimotor gating deficits in Brattleboro rats by acute administration of clozapine and a neurotensin agonist, but not haloperidol: a potential predictive model for novel antipsychotic effects.

Prepulse inhibition (PPI) of acoustic startle is decreased in unmedicated schizophrenia patients and similar deficits can be induced in rats through pharmacological, environmental, or neuroanatomical manipulations. Recently, we reported that Brattleboro (BB) rats, a Long Evans (LE) strain with a single gene mutation, have inherent deficits in PPI homologous to those observed in schizophrenia patients. We also reported that PPI deficits in BB rats could be reversed by chronic but not acute administration of 0.5 mg/kg haloperidol. No other dose or drug was tested in that experiment. In this study, we tested the effects of acute subcutaneous administration of several doses of haloperidol as well as the second-generation antipsychotic, clozapine, and the putative novel antipsychotic, PD149163, a neurotensin mimetic that crosses the blood-brain barrier. Consistent with our previous report, BB rats exhibited PPI deficits compared to LE rats and none of the doses of haloperidol produced a significant effect on this PPI deficit. In contrast, 10 and 15 mg/kg of clozapine and all the doses of PD149163 tested reversed the PPI deficits in BB rats. In addition, haloperidol, but not clozapine or PD149163 produced significant catalepsy in BB rats, supporting the notion that PD149163 has a profile consistent with atypical antipsychotics and providing support for the predictive validity of the PPI results. These results further strengthen the notion that the BB rat is a useful predictive model of antipsychotic efficacy and suggest that this model may differentiate between antipsychotics belonging to different therapeutic categories, for example, first- and second-generation agents.

Receptor trafficking via the perinuclear recycling compartment accompanied by cell division is necessary for permanent neurotensin cell sensitization and leads to chronic mitogen-activated protein kinase activation.

Most G protein-coupled receptors are internalized after interaction with their respective ligand, a process that subsequently contributes to cell desensitization, receptor endocytosis, trafficking, and finally cell resensitization. Although cellular mechanisms leading to cell desensitization have been widely studied, those responsible for cell resensitization are still poorly understood. We examined here the traffic of the high affinity neurotensin receptor (NT1 receptor) following prolonged exposure to high agonist concentration. Fluorescence and confocal microscopy of Chinese hamster ovary, human neuroblastoma (CHP 212), and murine neuroblastoma (N1E-115) cells expressing green fluorescent protein-tagged NT1 receptor revealed that under prolonged treatment with saturating concentrations of neurotensin (NT) agonist, NT1 receptor and NT transiently accumulated in the perinuclear recycling compartment (PNRC). During this cellular event, cell surface receptors remained markedly depleted as detected by both confocal microscopy and (125)I-NT binding assays. In dividing cells, we observed that following prolonged NT agonist stimulation, NT1 receptors were removed from the PNRC, accumulated in dispersed vesicles inside the cytoplasm, and subsequently reappeared at the cell surface. This NT binding recovery allowed for constant cell sensitization and led to a chronic activation of mitogen-activated protein kinases p42 and p44. Under these conditions, the constant activation of NT1 receptor generates an oncogenic regulation. These observations support the potent role for neuropeptides, such as NT, in cancer progression.

Clinically validated peptides as templates for de novo peptidomimetic drug design at G-protein-coupled receptors.

Recent advances in the development of potent and selective peptide and non-peptide ligands for peptidergic receptors are anticipated to help further unravel the roles of class I and II G-protein-coupled receptors in the pathogenesis of human diseases and to accelerate the clinical utility of small molecule peptidomimetics. Peptidomimetic drug discovery directed towards somatostatin agonists, urotensin II antagonists, gonadotropin-releasing hormone antagonists, neurotensin and complement C5a modulators, melanocortin-4 agonists and vasopressin V(2) agonists has achieved success through integration of conformational-based drug design, site-directed mutagenesis, screening, combinatorial chemistry and classical medicinal chemistry. Acceptance that discreet ensembles of secondary structural motifs underpin the interactions of peptides with their cognate receptors has enabled the development of molecules which mimic or stabilize such pharmacophores.

Characterization of beta-lactotensin, a bioactive peptide derived from bovine beta-lactoglobulin, as a neurotensin agonist.

beta-Lactotensin (beta-LT: His-Ile-Arg-Leu) is an ileum-contracting peptide derived from residues No. 146-149 of bovine beta-lactoglobulin. The ileum-contracting activity of beta-LT was blocked by the NT1 antagonist SR48692. beta-LT was selective for the neurotensin NT2 receptor while neurotensin was selective for the NT1 receptor. beta-LT is the first natural ligand showing selectivity for the NT2 receptor. beta-LT showed hypertensive activity after intravenous administration at a dose of 30 mg/kg in conscious rats, while neurotensin showed hypotensive activity. The hypertensive activity of beta-LT was blocked by levocabastine (1 mg/kg, i.v.), an NT2 antagonist. SR48692, which blocked the hypotensive activity of neurotensin, had no effect on the hypertensive activity of beta-LT. These results suggest that the hypertensive activity of beta-LT is mediated by the NT2 receptor. It was concluded that the NT1 and NT2 receptors mediate the opposite effect on blood pressure.

A systemically administered neurotensin agonist blocks disruption of prepulse inhibition produced by a serotonin-2A agonist.

Prepulse inhibition (PPI) of the startle reflex can be disrupted by drugs that act as agonists at the serotonin (5-HT) 2A receptor, such as DOI, and this effect is blocked by drugs that inhibit 5-HT2A transmission. We tested the effects of systemic administration of PD149163, a neurotensin agonist, on DOI-induced disruption of PPI in Sprague-Dawley rats. PD149163 completely and dose dependently blocked the PPI deficits produced by DOI. These findings suggest that, in addition to their established ability to inhibit dopamine transmission, neurotensin agonists may also inhibit 5-HT2A transmission, a pharmacological feature associated with atypical antipsychotic drugs.