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Background and Aims Chronic kidney disease–associated pruritus (CKD-aP) is a common and highly distressing condition in patients undergoing hemodialysis (HD). CKD-aP is associated with sleep disturbances, significant quality-of-life (QoL) impairment, and increased morbidity and mortality. Difelikefalin (DFK) is a selective kappa opioid receptor agonist in development for CKD-aP that has minimal central nervous system penetration. In placebo-controlled phase 3 trials of patients with moderate to severe CKD-aP undergoing HD, intravenous (IV) DFK had an acceptable safety profile and demonstrated significant reductions vs placebo in itch intensity. We report safety and effectiveness outcomes, including itch-related QoL and sleep measures, from a phase 3 open-label study of DFK in patients with moderate to severe CKD-aP. Method This multicenter, open-label study conducted in the United States and Europe enrolled patients with moderate to severe CKD-aP (mean baseline 24-hour Worst Itching Intensity Numerical Rating Scale [WI-NRS] score ≥5) undergoing HD for ≥3 months. Patients received IV DFK 0.5 mcg/kg 3 times/week at the end of each HD session for up to 12 weeks. Predefined effectiveness endpoints at week 12 included ≥3-point and ≥4-point improvement in the weekly mean of the 24-hour WI-NRS score (range from 0 [no itching] to 10 [worst itching imaginable]). Change from baseline in QoL at week 12 was assessed using the 5-D itch and Skindex-10, multidimensional itch-related questionnaires validated in CKD-aP (higher scores indicate worse QoL). The proportion of patients with no problems (score of 1) on the skin irritation and self-confidence domains of the EQ-PSO questionnaire was evaluated. Post hoc endpoints included complete resolution in WI-NRS (≥75% of week 12 scores 0 or 1) and Sleep Quality Questionnaire total score assessments (range of possible scores, 0 [did not interfere] to 10 [completely interfered]), including ≥3-point and ≥4-point improvement in weekly mean score and complete resolution (all scores of 0) at week 12. Safety assessments and adverse events (AEs) were evaluated. Data were summarized descriptively. Results Among 222 patients who received DFK, 197 (88.7%) completed the study. At baseline, mean ±SD age was 58.1 ±12.8 years and 54.5% of patients were male. Baseline mean ±SD WI-NRS score was 7.6 ±1.3, Sleep Quality score was 6.6 ±2.2, 5-D itch score was 17.1 ±3.5, and Skindex-10 score was 32.9 ±14.3. At week 12, the majority of patients achieved ≥3-point and ≥4-point improvement in WI-NRS (73.7% and 59.3%) and Sleep Quality score (66.0% and 56.7%). Complete resolution of WI-NRS and Sleep Quality score was observed in 29.4% and 19.1% of patients, respectively (Figure). DFK was associated with improvements in mean 5-D itch (−7.1 ±4.3) and Skindex-10 (−21.0 ±15.6) scores at week 12. The proportion of patients reporting no problems in the skin irritation EQ-PSO domain increased from 1.4% at baseline to 28.9% at week 12, and self-confidence EQ-PSO domain scores increased from 63.5% at baseline to 73.2% at week 12. Overall, 64.4% (143/222) of patients reported ≥1 treatment-emergent AE (TEAE). The most commonly reported TEAEs (≥4% of patients) were diarrhea (5.0% [11/222]), nausea (4.5% [10/222]), and hyperkalemia (4.1% [9/222]). Serious TEAEs were reported by 20.3% (45/222) of patients; no serious TEAEs were related to study drug. Conclusion In this phase 3 open-label study in patients with moderate to severe CKD-aP undergoing HD, DFK was generally well tolerated with an acceptable safety profile. DFK demonstrated effectiveness based on reduction of itch intensity, and improvements in sleep quality and itch-related QoL at week 12. The majority of patients reported ≥3-point or ≥4-point improvement in WI-NRS and Sleep Quality scores, with some reporting complete resolution. Findings from this open-label study provide insight into the potential real-world effectiveness of DFK in moderate to severe CKD-aP.

Difelikefalin is a peripherally restricted and selective agonist of kappa opioid receptors that are considered to be important in modulating pruritus in conditions such as chronic kidney disease.In this double-blind, placebo-controlled, phase 3 trial, we randomly assigned patients undergoing hemodialysis who had moderate-to-severe pruritus to receive either intravenous difelikefalin (at a dose of 0.5 μg per kilogram of body weight) or placebo three times per week for 12 weeks. The primary outcome was the percentage of patients with an improvement (decrease) of at least 3 points from baseline at week 12 in the weekly mean score on the 24-hour Worst Itching Intensity Numerical Rating Scale (WI-NRS; scores range from 0 to 10, with higher scores indicating greater itch intensity). The secondary outcomes included the change from baseline in itch-related quality-of-life measures, the percentage of patients with an improvement of at least 4 points in the WI-NRS score at week 12, and safety.A total of 378 patients underwent randomization. A total of 82 of 158 patients (51.9%) in the difelikefalin group had a decrease of at least 3 points in the WI-NRS score (primary outcome), as compared with 51 of 165 (30.9%) in the placebo group. The imputed percentage of patients with a decrease of at least 3 points in the WI-NRS score was 49.1% in the difelikefalin group, as compared with 27.9% in the placebo group (P0.001). Difelikefalin also resulted in a significant improvement from baseline to week 12 in itch-related quality of life as measured by the 5-D itch scale and the Skindex-10 scale. The imputed percentage of patients with a decrease of at least 4 points in the WI-NRS score at week 12 was significantly greater in the difelikefalin group than in the placebo group (37.1% [observed data: 64 of 158 patients] vs. 17.9% [observed data: 35 of 165 patients], P0.001). Diarrhea, dizziness, and vomiting were more common in the difelikefalin group than in the placebo group.Patients treated with difelikefalin had a significant reduction in itch intensity and improved itch-related quality of life as compared with those who received placebo. (Funded by Cara Therapeutics; KALM-1 ClinicalTrials.gov number, NCT03422653.).

Background Peripherally selective opioids may be beneficial in visceral pain management due to absence of centrally mediated side effects. The objectives of this study were: (1) to assess the effects of a peripherally selective tetrapeptide kappa-opioid receptor agonist, CR665, on experimental pain from multi-modal stimulation of skin, muscle, and viscera, and (2) contrast these effects with those of oxycodone (centrally acting opioid). Methods The study was designed as a single-center, single-dose, randomized, double-blind, placebo and active-controlled, double-dummy, three-way, crossover study in healthy males. Subjects received the following treatments in randomized order: (1) CR665 (0.36 mg/kg) administered intravenously over 1 h, (2) oxycodone (15 mg) administered orally, and (3) placebo administered intravenously and orally. The following pain tests were used: (1) cutaneous pinch pain tolerance threshold, (2) pressure pain detection and tolerance thresholds, (3) cuff pressure pain tolerance threshold, and (4) pain rating thresholds to distension and thermal stimulation of the esophagus. Measurements were performed before dosing and at 30, 60, and 90 min after dosing. Results Compared to placebo, oxycodone elevated cutaneous pinch pain tolerance (P < 0.001) and cuff pressure pain tolerance threshold (P < 0.001), as well as pain rating thresholds (visual analogue scale = 7) to esophageal distension (P < 0.001) and thermal stimulation (P < 0.002). Compared to placebo, CR665 significantly increased the pain rating threshold to esophageal distension (P < 0.005) but reduced the pain tolerance threshold to skin pinching (P = 0.007). Conclusion CR665 had a selective effect on visceral pain. Oxycodone exhibited a generalized effect, elevating thresholds for cutaneous, deep somatic, and visceral pain stimulation.

5-Hydroxytryptamine (5-HT)(2C) receptor agonists hold promise for the treatment of obesity. In this study, we describe the in vitro and in vivo characteristics of lorcaserin [(1R)-8-chloro-2,3,4,5-tetrahydro-1-methyl-1H-3 benzazepine], a selective, high affinity 5-HT(2C) full agonist. Lorcaserin bound to human and rat 5-HT(2C) receptors with high affinity (K(i) = 15 +/- 1 nM, 29 +/- 7 nM, respectively), and it was a full agonist for the human 5-HT(2C) receptor in a functional inositol phosphate accumulation assay, with 18- and 104-fold selectivity over 5-HT(2A) and 5-HT(2B) receptors, respectively. Lorcaserin was also highly selective for human 5-HT(2C) over other human 5-HT receptors (5-HT(1A), 5-HT(3), 5-HT(4C), 5-HT5(5A), 5-HT(6), and 5-HT(7)), in addition to a panel of 67 other G protein-coupled receptors and ion channels. Lorcaserin did not compete for binding of ligands to serotonin, dopamine, and norepinephrine transporters, and it did not alter their function in vitro. Behavioral observations indicated that unlike the 5-HT(2A) agonist (+/-)-1-(2,5-dimethoxy-4-phenyl)-2-aminopropane, lorcaserin did not induce behavioral changes indicative of functional 5-HT(2A) agonist activity. Acutely, lorcaserin reduced food intake in rats, an effect that was reversed by pretreatment with the 5-HT(2C)-selective antagonist 6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy)pyridin-3-yl-carbamoyl]indoline (SB242,084) but not the 5-HT(2A) antagonist (R)-(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methanol (MDL 100,907), demonstrating mediation by the 5-HT(2C) receptor. Chronic daily treatment with lorcaserin to rats maintained on a high fat diet produced dose-dependent reductions in food intake and body weight gain that were maintained during the 4-week study. Upon discontinuation, body weight returned to control levels. These data demonstrate lorcaserin to be a potent, selective, and efficacious agonist of the 5-HT(2C) receptor, with potential for the treatment of obesity.

Monkeys that receive chronic low dose (CLD) 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration develop deficits in spatial delayed-response task performance. The present study examined the extent to which SIB-1553A [(±)-4-{[2-(1-methyl-2-pyrrolidinyl)ethyl]thio}phenol hydrochloride], a novel neuronal nicotinic acetylcholine receptor (nAChR) agonist with selectivity for β4 subunit-containing nAChRs, could counteract this cognitive deficit produced by CLD MPTP exposure. Prior to MPTP treatment, monkeys displayed a delay-dependent decrement in performance on a variable delayed response task. CLD MPTP treatment caused a shift to a delay-independent pattern of responding on this task, such that short-delay trials were performed as poorly as long-delay trials. At lower doses (e.g., 0.025 mg/kg), SIB-1553A significantly improved performance on short-delay trials but only at 24 h after drug administration. At higher doses (e.g., 0.50 mg/kg), SIB-1553A significantly improved performance on both short- and long-delay trials at both 20 min and 24 h after drug administration. When tested 24 h after drug administration, monkeys performed long-delay trials with greater accuracy than they did under normal (pre-MPTP) conditions. These results suggest that at lower doses, SIB-1553A may be more effective in improving attentional deficits associated with CLD MPTP exposure, whereas at higher doses, SIB-1553A may effectively improve both attentional and memory performance.

The potential antiparkinsonian effects of the centrally acting, subtype-selective neuronal nicotinic acetylcholine receptor agonist (S)-(7-)--5-ethynyl-3-(1-methyl-2-pyrrolidinyl)-pyridine (SIB-1508Y) was assessed on motor symptoms and disability scale ratings in three monkeys previously made parkinsonian by chronic exposure to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Compared with levodopa (L-dopa), SIB-1508Y exerted only mild antiparkinsonian effects when administered alone. Emetic effects of this drug interfered with potential therapeutic effects at higher doses. However, when a low, ineffective dose of SIB-1508Y was combined with low, ineffective doses of L-dopa, a significant clinical effect was observed. These data suggest that subtype-selective nicotinic acetylcholine receptor agonists may hold promise as antiparkinsonian agents, and when administered in combination with L-dopa may allow a reduction in the dose of L-dopa needed to achieve a significant clinical effect.

Thymopentin, a synthetic pentapeptide (Arg-Lys-Asp-Val-Tyr) corresponding to amino acids 32-36 of the thymic polypeptide thymopoietin, has been reported to block adrenocorticotrope responses to stress. The purpose of the present study was to explore potential antistress properties of a synthetic analogue of thymopentin, IRI-514 (Ac-Arg-Pro-Asp-Phe-NH2) using a behavioral response to a stressor. The behavioral response to social conflict stress (resident-intruder paradigm) was evaluated by the elevated plus-maze test of anxiety in adult Wistar rats. A single subcutaneous (SC) administration of IRI-514, 48 h before stress, dose-dependently reversed the anxiety-like behavior induced by the social stress. The effect of IRI-514 was present over an extended period (24-72 h) following SC administration and was maximally effective at a dose of 1 mg/kg. These results indicate that IRI-514 has a long-lasting modulatory effect on behavioral responses to a stressor, and suggest that thymopoietin-derived peptides may have a role in modulating both behavioral and neuroendocrine responses to stress.

CRF in the central nervous system appears to hve activating properties on behavior and to coordinate behavioral responses to stressors. These behavioral effects of CRF appear to be independent of the pituitary-adrenal axis and can be reversed by CRF antagonists. CRF antagonist administration reverses not only decreases in behavior associated with stress, but also increases in behavior associated with stress, thus suggesting that the role of CRF is stress dependent and not intrinsic to a given behavioral response. Further, microinjection of alpha-helical CRF 9-41 and immunotargeting of CRF neurons in separate brain compartments reveal a link between the anatomical sites that contain CRF and the nature of the behavioral response to stressors that can be modified by suppression of endogenous CRF activity therein. These actions of CRF in coordinating coping responses to stress at several bodily levels are consistent with a role for CRF similar to the dual role of other hypothalamic releasing factors in integrating hormonal and neural mechanisms by acting both as secretagogues for anterior pituitary hormones and as extrapituitary peptide neurotransmitters. Moreover, dysfunction in such a fundamental homeostatic system may be the key to a variety of pathophysiological conditions including mental disorders.

Previous work has shown that administration of corticotropin-releasing factor (CRF) into the lateral ventricle antagonizes the orexigenic effect of neuropeptide Y (NPY), and central injection of CRF antagonist, α-helical CRF(9–41) enhanced NPY-induced food intake in satiated rats. The aim of the present study was to determine the effects of selective inactivation of hypothalamic CRF neurons on food intake induced by NPY injection and to delinate which hypothalamic nucleus is involved in this NPY/CRF interaction related to the regulation of food intake. Impairment of CRF neuron function by immunotargeting of a ricin A chain toxin with a monoclonal antibody to CRF (CRF-MAb) has been previously reported. Administration of CRF-MAb/toxins into the paraventricular nucleus (PVN) two weeks prior to testing produced markedly enhanced eating induced by injection of NPY into the same nucleus. This effect was accompanied by a 60% decrease in CRF content within the hypothalamus and a 43% decrease of CRF in the median eminence, a site of projection of CRF neurons from the PVN. In contrast, injection of CRF-MAb/toxins into the ventromedial nucleus of the hypothalamus (VMH) did not modify the feeding induced by NPY injection into this hypothalamic area. Systemic pretreatment with the synthetic glucocorticoid dexamethasone at a dose known to downregulate the levels of CRF in the PVN also enhanced the feeding induced by intra-PVN injection of NPY.This suggests that an equilibrium between CRF and NPY neuronal function within the PVN may play an important role in the regulation of food intake. This interactive mechanism may provide some partial explanation of the eating disorders related to stress, in particular anorexia nervosa.

Evidence from animal and human studies suggests that neuropeptide Y (NPY) may be a potent endogenous anxiolytic. The anatomic structures mediating this action of the peptide remain unknown. Furthermore, in addition to its anxiolytic-like effects, intracerebroventricular administration of NPY induces food intake through hypothalamic mechanisms, making the anxiolytic-like action of the peptide more difficult to interpret. The purpose of this study was to examine the anatomic substrate for the effects of NPY on anxiety, and to characterize the NPY receptors mediating these effects. Intracerebroventricular injection of NPY produced increased food intake in free-feeding animals, and dose-dependent anticonflict/anxiolytic-like effects in an established animal model of anxiety, the Geller-Seifter punished responding test. In contrast, microinjection of NPY into the central nucleus of the amygdala did not increase food intake in free-feeding animals, did not affect unpunished lever pressing for food, but did reproduce the anticonflict/anxiolytic-like effect with high potency. The selective NPY-Y1 agonist, p[Leu31,Pro34]NPY was approximately equipotent with native NPY in the conflict paradigm, and markedly more potent than the Y2 agonist, NPY13-36. Intrastriatal injections had no effect on conflict behavior. Thus, activation of Y1 receptors in the central nucleus of the amygdala produces effects similar to established anxiolytics without affecting food intake, suggesting that Y1-receptors in the amygdala may be a substrate for anxiolytic actions of NPY.

We previously demonstrated that cellular toxins added to a cytotoxic IgG2a monoclonal antibody to corticotropin releasing factor (CRF-MAb) may specifically penetrate some hypothalamic CRF neurons, after central injection near the paraventricular nuclei. We attempt here to evaluate the consequential effects on the CRF neurons functioning. Such a toxic mix, 4 weeks after its central injection, caused a marked reduction (66%) of the chronic adrenocorticotropic hormone (ACTH) release in response to a bilateral adrenalectomy (7th day). This change was accompanied by a reduction in the CRF concentration (43%) measured in the median eminence. We concluded that specific internalization of toxins, by the way of CRF-MAb, leads to a long-term dysregulation of the CRF synthesis and/or neuronal transport.

Peripherally selective opioids may be beneficial in visceral pain management due to absence of centrally mediated side effects. The objectives of this study were: (1) to assess the effects of a peripherally selective tetrapeptide kappa-opioid receptor agonist, CR665, on experimental pain from multi-modal stimulation of skin, muscle, and viscera, and (2) contrast these effects with those of oxycodone (centrally acting opioid).The study was designed as a single-center, single-dose, randomized, double-blind, placebo and active-controlled, double-dummy, three-way, crossover study in healthy males. Subjects received the following treatments in randomized order: (1) CR665 (0.36 mg/kg) administered intravenously over 1 h, (2) oxycodone (15 mg) administered orally, and (3) placebo administered intravenously and orally. The following pain tests were used: (1) cutaneous pinch pain tolerance threshold, (2) pressure pain detection and tolerance thresholds, (3) cuff pressure pain tolerance threshold, and (4) pain rating thresholds to distension and thermal stimulation of the esophagus. Measurements were performed before dosing and at 30, 60, and 90 min after dosing.Compared to placebo, oxycodone elevated cutaneous pinch pain tolerance (P0.001) and cuff pressure pain tolerance threshold (P0.001), as well as pain rating thresholds (visual analogue scale = 7) to esophageal distension (P0.001) and thermal stimulation (P0.002). Compared to placebo, CR665 significantly increased the pain rating threshold to esophageal distension (P0.005) but reduced the pain tolerance threshold to skin pinching (P = 0.007).CR665 had a selective effect on visceral pain. Oxycodone exhibited a generalized effect, elevating thresholds for cutaneous, deep somatic, and visceral pain stimulation.

Previous studies have shown that microinjection of cytotoxic lgG2a monoclonal antibody to rat/human corticotropin-releasing factor (CRF-MAb) into the hypothalamic paraventricular nucleus of Long Evans rats resulted in antibody uptake into specific neurons of the paraventricular nucleus. We tested the hypothesis that this neuronal uptake may allow non-linked toxins to enter into specific neurons. The effects of central or peripheral administration of a mix containing CRF-MAb and cellular toxins (toxin/ CRF-MAb) on plasma adrenocorticotropin levels were determined before and after exposure to ether stress in freely moving rats. Peripheral injection (jugular vein) of the toxin/CRF-MAb mix or injection into the supraoptic nuclei did not affect resting or stress-induced adrenocorticotropin secretion. In contrast, bilateral injection of the same mix into the paraventricular nucleus or the lateral ventricle caused a consistent 70% reduction of the ether stress-induced adrenocorticotropin release but had no significant effect on the resting adrenocorticotropin levels. The blocking effect of intracerebroventricular administration disappeared after 24 h whereas the blockade persisted for at least 15 days after local injection into the paraventricular nucleus. The injection into the paraventricular nucleus of CRF-MAb without toxins restricted the inhibitory effects to 24 h. These data suggest that administration of a mix of toxins added to a specific lgG2a monoclonal antibody to CRF resulted in a long-term interference with the control of adrenocorticotropin secretion. This new approach may be of use for elucidating the physiological roles of different central peptidergic systems.

Previous immunocytochemical studies reported that when specific monoclonal antibody directed against vasopressin (VP) (VP-MAb) was injected in vivo above the rat hypothalamic nuclei, it penetrated and was specifically transported by VP-producing neurons. In this study, using the same methodological approach, the fate of monoclonal antibody directed against corticotropin-releasing factor (CRF) (CRF-MAb) injected in vivo above the paraventricular nucleus (PVN) of the rat brain was investigated by immunocytochemistry in male Zucker rats and adrenalectomized or colchicine-pretreated malo Long-Evans rats. The simultaneous immunocytochemical localization of the injected CRF-MAb and endogenous peptides and enzyme synthesized by the neurons penetrated by the antibody, demonstrated that CRF-MAb was mainly detected in CRF neurons. But the CRF-MAb was also detected in VP, oxytocin, neuropeptide Y and tyrosine hydroxylase-producing neurons of the PVN. CRF-MAb was therefore localized in PVN neurons which synthesize CRF and in PVN neurons with physiological and morphological relationships with the CRF peptidergic system. Before obtaining biological effects of injected CRF-MAb, the results described here suggest that specific monoclonal antibodies provide a useful specific tool for elucidating the functional relationship between neuronal systems.

Corticotropin-releasing factor (CRF), when administered directly into the CNS, can have activating properties on behaviour and can enhance behavioural responses to stress. CRF injected intraventricularly produces a dose-dependent increase in locomotor activity and increased responsiveness to an acoustic startle stimulus. However, this profile of activation changes to enhanced suppression of behaviour in stressful situations and includes increased freezing, increased conditioned suppression, increased conflict, decreased feeding and decreased behaviour in a novel open field. These effects of CRF are independent of the pituitary-adrenal axis and can be reversed by the CRF antagonist alpha-helical CRF(9-41). More importantly, the CRF antagonist can also reverse many behavioural responses to stressors. alpha-Helical CRF(9-41) reverses stress-induced fighting behaviour, stress-induced freezing, stress-induced suppression of feeding, stress-induced decreases in exploration of an elevated plus maze, fear-potentiated startle and the development of conditioned suppression. Intracerebral microinjections suggest that the amygdala may be an important site for the anti-stress effects of alpha-helical CRF(9-41). These results suggest that endogenous CRF systems in the CNS may have a role in mediating behavioural responses to stress and further suggest that CRF in the brain may function as a fundamental behavioural activating system. This CRF system may be particularly important in situations where an organism must mobilize not only the pituitary-adrenal system but also the CNS in response to environmental challenge.

Kappa-(kappa) opioid receptors are widely distributed in the periphery and activation results in antinociception; however supraspinal acting kappa-agonists result in unwanted side effects. Two novel, all d-amino acid, tetrapeptide kappa-opioid receptor agonists, FE 200665 and FE 200666, were identified and compared to brain penetrating (enadoline) and peripherally selective (asimadoline) kappa-agonists as potential analgesics lacking unwanted central nervous system (CNS) side effects. In vitro characterization was performed using radioligand binding and GTP gamma S binding. Antinociception was evaluated in both mice and rats. Rotarod tests were performed to determine motor impairment effects of the kappa-agonists. FE 200665 and FE 200666 showed high affinity for human kappa-opioid receptor 1 (Ki of 0.24 nM and 0.08 nM, respectively) and selectivity for human kappa-opioid receptor 1 (human kappa-opioid receptor 1/human mu-opioid receptor/human delta-opioid receptor selectivity ratios of 1/16,900/84,600 and 1/88,600/1,250,000, respectively). Both compounds demonstrated agonist activity in the human kappa-opioid receptor 1 [35S]GTP gamma S binding assay (EC50 of 0.08 nM and 0.03 nM) and resulted in dose-related antinociception in the mouse writhing test (A50: 0.007 and 0.013 mg/kg, i.v., respectively). Markedly higher doses of FE 200665 and FE 200666 were required to induce centrally-mediated effects in the rotarod assay (548- and 182-fold higher doses, respectively), and antinociception determined in the mouse tail-flick assay (1429- and 430-fold fold higher doses, respectively) after peripheral administration supporting a peripheral site of action. The potency ratios between central and peripheral activity suggest a therapeutic window significantly higher than previous kappa-agonists. Furthermore, FE 200665 has entered into clinical trials with great promise as a novel analgesic lacking unwanted side effects seen with current therapeutics.

Rationale: Epidemiological studies of smokers suggest that there is a link between nicotine and depression. Nonetheless, few studies have examined the potential use of nicotinic ligands in the treatment of depression. Objectives: The goal of this study was to evaluate the effects of SIB-1508Y, a novel subtype-selective ligand for high affinity nicotinic acetylcholine receptors (nAChRs), in the learned helplessness model of depression in rats. Methods: In this model, exposure to inescapable footshock produces a lasting deficit in escape responses emitted in a subsequent conditioned avoidance procedure (learned helplessness). The effect of SIB-1508Y on learned helplessness was compared to the clinically used antidepressants, imipramine and fluoxetine, and the non-selective nAChR ligand, nicotine. Results: Similarly to imipramine and fluoxetine, subchronic treatment (5 days) with SIB-1508Y reversed the escape deficit in the learned helplessness model in a dose dependent manner. The effect of SIB-1508Y on learned helplessness was still apparent 1 week following drug administration and was also maintained after 4 weeks of daily administration. In contrast, while nicotine was able to attenuate the learned helplessness deficit, this trend only reached statistical significance after chronic administration. The non-competitive ion channel blocker mecamylamine increased escape failures when administered alone and blocked the effects of SIB-1508Y but not imipramine. SIB-1508Y also produced an increase in avoidance responding, which suggests an enhancement of learning. Conclusion: These results not only suggest a role for nAChRs in the development of a depressive-like syndrome, but also that subtype-selective nAChR agonists, such as SIB-1508Y, may offer a novel therapeutic approach to the treatment of depression.

A series of conformationally restricted analogues of nicotine has been synthesized and evaluated as agonists of neuronal acetylcholine receptors. Compound 2 (SIB-1663), which selectively activated human recombinant alpha 2 beta 4 and alpha 4 beta 4 nAChRs, was shown to be active in animal models of Parkinson's disease and pain.

Publisher Summary This chapter critically reviews evidence of physiologically relevant appetite stimulation by neuropeptide Y (NPY) and to forecast how this knowledge might be applied in the context of eating disorders. Recently, a novel receptor for NPY, termed “Y5”, has been cloned from rat hypothalamus and appears to meet all the criteria necessary to be designated the “feeding” receptor distinct from previously cloned Y-type receptors. Striking appetite stimulation by NPY receptor agonists under pharmacological conditions can only elucidate the true physiological role of this neuropeptide if NPY receptor antagonists are found to be equally effective in blunting feeding produced under naturalistic conditions. Evidence for insulin actions on NPY gene expression was provided by studies that demonstrated that central infusion of insulin could depress elevated preproNPY mRNA levels induced by food deprivation of lean Zucker rats but was without effect on preproNPY mRNA content in fed or fasted obese Zucker rats. The extrapolation of the NPY-induced overeating and overweight syndrome in rodents to human obesity must await knowledge about the role of NPY in the human brain.

In summary, endogenous CRF has been demonstrated to play an important role in the endocrine but also autonomic and behavioral responses to a stressor and to mediate some of the signs and symptoms observed in human affective and anxiety disorders. These findings led to the hypothesis that the anxiety that characterizes drug withdrawal, such as ethanol withdrawal in humans, may be related in part to the action of CRF-producing neurons in the CNS. Indeed, rats made dependent on an ethanol liquid diet showed significant signs of enhanced stress responsiveness that was blocked by intracerebral administration of a CRF antagonist. At this time little is known about the specific site of action for endogenous CRF. However, recent studies using local administration of CRF antagonist and in vivo CRF microdialysis suggest that the central nucleus of the amygdala may be an important site for the increases in CRF activity associated with the anxiogenic effects of ethanol withdrawal. Although preliminary, these results propound that ethanol dependence may involve a prolonged dysregulation of the CRF system in the basal forebrain that may contribute to the increased motivational effect of ethanol withdrawal.

Central administration of a Corticotropin-Releasing Factor (CRF) antagonist is well documented to attenuate a variety of behavioral responses to several distinct stressors; however, it is not yet clear whether the activation of CRF neurons is dependent on the type or intensity of the experimental stressor, or rather on the particular behavioral response to stress under study. To test the generality of the stress-protective effect of the CRF antagonist, alpha-helical CRF9-41, (1, 5 or 25 micrograms intracerebroventricularly), the present experiments employed a sensitive index of anxiogenic-like behavior by measuring suppression in exploration on the elevated plus-maze following exposure to social, swim, or restraint stressors. A 1 but not 5 or 25 micrograms dose of the CRF antagonist administered just prior to social, swim, or restraint stress reversed the stress-induced inhibition of exploratory behavior. Chlordiazepoxide and the steroid anesthetic, alphaxalone, also attenuated the anxiogenic-like effect of restraint stress and elevated the baseline exploratory behavior of nonstressed control groups. Although the stressors produced a graded secretion of adrenocorticotropin (ACTH) with the ranking restraintswimsocial, the relative amplitude of behavioral reactivity to social, swim, and restraint stress was comparable. The relative efficacy of the CRF antagonist to reverse the stressor effects was also comparable. These results suggest that antagonism of activated brain CRF systems attenuates the behavioral response to stress regardless of the type or intensity of the stressor as measured by ACTH secretion.

Procedures were developed to permit the measurement of corticotropin-releasing factor in perfusate collected from microdialysis probes implanted in various brain areas of anesthetized and awake rats. Initially in vitro experiments were carried out to optimize the recovery of corticotropin-releasing factor and the radioimmunoassay conditions. Addition of a specific antiserum against corticotropin-releasing factor to the perfusion medium (artificial cerebrospinal fluid) increased the relative in vitro recovery over a range of different flow rates (1-10 microliters/min) using commercially available microdialysis probes with a membrane cutoff of 20,000 mol. wt. This procedure increased recovery from 3% to 6% at flow rate of 2.5 microliters/min, and from 4% to 8% at a flow rate of 5 microliters/min. In vivo experiments were performed with a flow rate of 3.3 microliters/min and 50-microliters fractions were used for radioimmunoassay. In each experiment, the standard curve of the radioimmunoassay was constructed from aliquots of the same medium used to perfuse the probe. Basal levels of corticotropin-releasing factor in dialysate collected from the mediobasal hypothalamus of anesthetized rats were estimated to be 0.75 +/- 0.07 fmol/50 microliters. Raising the concentration of potassium (60 mM) in the perfusate increased corticotropin-releasing factor levels to 2.04 +/- 0.37 fmol/50 microliters. Hypertonic stress induced by intraperitoneal injection of 1.5M NaCl (20 ml/kg) elevated the levels to 1.32 +/- 0.07 fmol/50 microliters. A marked increase of corticotropin-releasing factor levels was also produced by a 10-min pulse of the potassium-channel blocker 4-aminopyridine (10 mM) included in the perfusate. A second stimulation pulse with 4-aminopyridine, administered 2 h after the first pulse again increased the levels, with a mean ratio between the first and second pulse of 0.97. Corticotropin-releasing factor efflux produced by the second stimulation pulse was completely inhibited by perfusion with calcium-free medium containing calcium-chelating agent ethyleneglycol tetraacetic acid (10 mM). In separate experiments, microdialysis probes were implanted in several brain areas of anesthetized rats. Basal and potassium-evoked levels of corticotropin-releasing factor were measured in dialysate collected from the amygdala (1.20 +/- 0.22 and 2.05 +/- 0.48 fmol/50 microliters, respectively) and frontal cortex (0.51 +/- 0.10 and 1.64 +/- 0.15 fmol/50 microliters, respectively). Corticotropin-releasing factor levels in the dorsal part of the third ventricle and in the striatum were below the detection limits. In awake rats, corticotropin-releasing factor levels in the mediobasal hypothalamus were 0.98 +/- 0.03 fmol/50 microliters.(ABSTRACT TRUNCATED AT 400 WORDS)

Central administration of neuropeptide Y (NPY) exerts a potent orexigenic effect in rats, whereas injection of corticotropin-releasing factor (CRF) suppresses food intake. Anatomical evidence of NPY-containing terminals located in close proximity to CRF-containing neurons and terminals of the hypothalamus and amygdala suggests possible interactions of these neuropeptide systems in food-intake regulation. The present study examined the effect of local administration of the CRF antagonist, alpha-helical CRF9-41, or peripheral treatment with dexamethasone on NPY-induced hyperphagia. Injection of a 250-ng dose of alpha-hel CRF within the paraventricular nucleus (PVN) of the hypothalamus significantly potentiated the feeding induced by a 500-ng dose of NPY injected into the same locus. In contrast, feeding induced by administration of the 500-ng dose of NPY into the ventromedial hypothalamus (VMH) was not modified by intra-VMH pre-treatment with a 250-ng dose of CRF antagonist. No effects of NPY or alpha-hel CRF on feeding were observed after administration into the central nucleus of the amygdala. Systemic pre-treatment with the synthetic glucocorticoid dexamethasone at a dose known to downregulate the function of CRF neurons in the PVN (100 micrograms/kg) enhanced feeding induced by intra-PVN administration of a 500-ng dose of NPY. These results suggest that hypothalamic CRF systems in the PVN exert inhibitory control over NPY-induced food intake.

We have previously demonstrated that vasopressin-producing neurons are the target of monoclonal antibodies to vasopressin microinjected into the brain tissue. At the same time, this central microinjection of vasopressin-monoclonal antibody into the supraoptic nuclei produced hydro-osmotic disorders mimicking the effects of a central diabetes insipidus. In order to investigate the increase in both duration and amplitude of the biological effects seen after the injection of vasopressin-monoclonal antibody, an immunoconjugate was constructed with the vasopressin-monoclonal antibody IgG1k isotype and the cytotoxic part of the ricin molecule, the ricin A chain. The biological parameters, such as diuresis and urine osmolality which are directly regulated by vasopressin, and vasopressin excretion, were measured after the central injection of this immunotoxin/immunoconjugate. The consequences of immunotoxin injection were also studied when immunotoxin was co-injected with monensin (50 nM) which has been shown to decrease the intracellular degradation of immunotoxin, and plasma complement, which has been shown to increase the neuronal uptake of immunotoxin. Single injection of immunotoxin near the hypothalamic supraoptic nuclei significantly increased diuresis and decreased vasopressin excretion. However, these effects were only transient and disappeared 24 h later. Four successive injections of immunotoxin (one per day) with monensin induced a decrease of vasopressin excretion which was still observed after a resting period of four days after the fourth injection. The long-term reduction of vasopressin excretion was induced in rats receiving four successive injections of a mixture consisting of immunotoxin with monensin and plasma complement. In such experiments, the vasopressin content of urine remained low (55% under the baseline value), two weeks after the fourth injection of immunotoxin. At the same time, the diuresis was increased (80% above the baseline value) and urine osmolality lowered (45% under the baseline value). When non-specific IgG replaced specific antibody, vasopressin excretion, diuresis as well as urine osmolality were unchanged. The results of this study demonstrated that the use of a specific immunotoxin results in a local interference with the vasopressinergic neurons and induces a long-term reduction of vasopressin secretion.

Previous work has characterized an anorexic action for endogenous, central nervous system corticotropin-releasing factor (CRF). Central injection of CRF decreases food intake induced pharmacologically by various appetite stimulants and a CRF antagonist attenuates restraint stress anorexia. Also, stressful physiological stimuli that are relevant to ingestive regulation, such as glucoprivation and protein nutrient deficiency, activate CRF systems. The present experiments examined the effects of exogenously administered CRF and a CRF antagonist, alpha-helical CRF(9-41), on spontaneous feeding induced by neuropeptide Y (NPY) and by a tail-pinch stressor. Pretreatment with a low dose of the CRF antagonist (1 microgram ICV) enhanced the hyperphagia induced by NPY while reducing the latency to begin feeding and increasing the duration of eating during tail pinch. Higher doses of alpha-hel CRF (5 and 25 micrograms ICV) exhibited diminishing or opposite effects. In contrast, CRF pretreatment (0.02, 0.1, and 0.5 microgram ICV) blocked the acquisition of tail-pinch feeding. Hence, while CRF administration impairs intake in these and other feeding paradigms, alpha-hel CRF actually facilitated dose dependently the intensity of the feeding response to NPY and tail pinch. These results suggest that endogenous CRF systems may play a role in modulating excessive feeding under conditions of evoked appetite and that brain CRF systems regulate feeding when excessive intake threatens to compromise the performance of other noningestive behaviors.