Synthesis and relative potencies of new constrained CRF antagonists

Two series of CRF antagonists with N alpha- and C alpha-methylated alanine and leucines were evaluated for their biological activities in vitro and in vivo in several systems. The poly-N-methylated analogue of alpha-helical-CRF9-41, [N alpha MeLeu10,15,27,37,N alpha MeAla22,32,41]-alpha-Hel-CRF9-41, was found to be considerably less potent than the parent non-N-methylated analogue. This result was expected on the basis that alpha-helicity was thought to be required for biological activity and the prediction that backbone substitutions on the nitrogen have a tendency to break alpha-helices (a hypothesis that was confirmed by circular dichroism). Next, a series of constrained analogues of the potent CRF antagonist, [DPhe12,Nle21,38]h/rCRF12-41, was synthesized that contained C alpha-methylleucine and/or C alpha-methylalanine (Aib) residues at selected positions. Because C alpha-methylation is recognized to increase alpha-helicity, and because there is now strong NMR data suggesting that residues 6-36 assume a well-defined alpha-helix, it was expected that these analogues would be more potent. Although usual solid-phase peptide synthesis procedures were followed, success in coupling the C alpha-methyl amino acids was obtained only with a 1:1 mixture of BOP/HOBt. In vitro potencies of the synthesized compounds were measured in a collagenase-dispersed anterior pituitary cell culture bioassay. Monosubstituted analogues were shown to be twice to one fourth as potent as the parent compound; while the pluri-substituted peptides were slightly less potent. This decrease in potency might be correlated to an unexpected lower helical content of the pluri-substituted compounds (as determined by CD spectroscopy), as it was suggested that the bioactive conformation of the CRF was predominantly alpha-helical. Interestingly, one analogue, [DPhe12,Nle21,38,C alpha-MeLeu37]h/rCRF12-41, was found to be more potent and longer acting than the parent compound in two in vivo assays measuring ACTH release after intravenous administration to adrenalectomized rats and reversal of stress-induced delay in gastric emptying in the rat after intracisternal administration. The molecular basis for this increased duration of action and potency is being investigated.     

Central CRF receptor antagonist α-helical CRF9-41 blocks reinstatement of extinguished fear: The role of the bed nucleus of the stria terminalis.

The present experiments assessed the necessity of central CRF in reinstatement of extinguished fear. Using the fear-potentiated startle procedure, rats were given light-shock pairings (fear conditioning) followed by light-alone extinction training. Rats were then given unsignaled shocks to reinstate fear to the light conditioned stimulus (CS). Intracerebroventricular administration of the CRF antagonist α-Helical CRF9-41 prior to reinstatement training dose-dependently prevented reinstatement. Further, α-Helical CRF9-41 administration prior to reinstatement training or the test for reinstatement of fear to the extinguished CS prevented reinstatement at both treatment times, suggesting that CRF activity is critical for this type of return of fear to an extinguished CS. The abolition of reinstatement by drug administration was not due to state-dependent learning, as rats treated with the drug prior to both reinstatement training or testing also failed α-Helical CRF9-41 in the bed nucleus of the stria terminalis suggested that this area is a site at which central CRF is involved in this form of relapse. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Adaptation to ozone in rats and its association with ascorbic acid in the lung

The present study evaluated the modulatory role of central corticotropin-releasing factor (CRF) systems in the mediation of the effects of acute exposure to the brain cannabinoid receptor agonist HU-210 [3-(1,1-dimethylheptyl)-(-)-11-hydroxy-delta 8-tetrahydrocannabinol] on defensive withdrawal behavior in male rats. The apparatus used for the defensive withdrawal test consisted of a small chamber, set on one side of a one-square meter open field. The actions of the potent CRF antagonist [D-Phe12,Nle21,38,C alpha MeLeu37]CRF (D-Phe CRF12-41) were examined on defensive behavior under both novel and familiar conditions. The acute i.c.v. administration of D-Phe CRF12-41 (0.2-5 micrograms/injection) antagonized the defensive behavior response to stressing conditions such as novelty or swim stress in field-habituated animals. The acute i.p. administration of HU-210 (4, 20 and 100 micrograms/kg) produced a clear dose-dependent stress-like effects in field-habituated animals, as reflected in the HU-210-induced increase in both the emergence latency and the mean time spent in the small chamber. The i.c.v. administration of 5 micrograms of D-Phe CRF12-41, 5 min before the administration of the cannabinoid prevented the stressing actions of HU-210 (20 micrograms/kg, but not 100 micrograms/kg). Acute administration of HU-210 also induced a dose-dependent increase in plasma corticosterone levels which was not antagonized by pretreatment with 5 micrograms of D-Phe CRF12-41. The present study suggests a role of central CRF systems in the mediation of the anxiogenic effects of brain cannabinoid receptor agonists. This finding is consistent with a direct hypothalamic effect of cannabinoids on the activation of the pituitary-adrenal axis.     

Effect of alpha-helical-CRF[9-41] on feeding in goldfish: involvement of cortisol and catecholamines

The anoretic effect of corticotropin-releasing factor (CRF) was not dependent on adrenal activation in goldfish (Carassius auratus). Moreover, an interaction between CRF and the hypothalamic catecholaminergic system in the central regulation of food intake was observed. The intracerebroventricular (icv) administration of CRF increased cortisol levels and reduced food intake and hypothalamic norepinephrine and dopamine content at 2 hr postinjection, with these effects reversed by alpha-helical CRF[9-41] pretreatment. The anoretic effect of CRF was independent of the circulating cortisol increase, because it was only evoked after icv injections but not after intraperitoneal (ip) administration. Furthermore, the increase in plasma cortisol levels induced by ip administration of this steroid did not modify feeding.     

Effects of corticotropin-releasing factor on circadian locomotor rhythm in the golden hamster

Stress produces a reduction in the amplitude of some circadian rhythms. The neurochemical mechanisms underlying stress-induced changes in circadian rhythms are not known. To investigate a possible role of corticotropin-releasing factor (CRF) in this phenomenon, three related experiments were carried out: activity rhythms of male golden hamsters (10/14 hours light/dark entrained, lights on at 0800 h) were measured 1) following the intracerebroventricular administration of CRF (0.5, 1.0, 2.0, or 4.0 microg) at two different times of day, 2) following social stress (30-min resident-intruder confrontation), 3) and following the administration of the CRF-antagonist alpha-helical CRF9-41 (2.0 microg) prior to a 15-min resident-intruder confrontation. CRF produced a significant, dose-related decrease in circadian rhythm amplitude following administration in the morning hours, but not in the afternoon. CRF also induced transient increases in activity post injection concomitant with an activation of the hypothalamic-pituitary-adrenocortical (HPA) system. Stress similarly reduced the amplitude of activity patterns and stimulated the HPA system. The stress-induced depression of circadian rhythm amplitude was significantly attenuated following alpha-helical CRF9-41. These data suggest a role for CRF in the stress-related modulation of circadian locomotor rhythm amplitude.     

Sleep and EEG power spectrum effects of the 5-HT1A antagonist NAN-190 alone and in combination with citalopram

The sleep and waking and EEG power spectrum effects of the putative 5-HT1A antagonist NAN-190 (0.5 mg/kg, i.p.) were studied alone and in co-administration with the selective serotonin re-uptake inhibitor citalopram (5.0 mg/kg, i.p.) in the rat. Citalopram, as in a prior dose-response study, reduced REM sleep. In addition, a slight increase in NREM sleep was observed. Citalopram reduced NREM fronto-parietal (FP) EEG power density in the 5-20 Hz range. When administered alone, NAN-190 suppressed REM sleep in the first 2 h, and reduced SWS-2 in the first 4 after administration. NAN-190 also suppressed selectively NREM sleep slow-wave activity in both fronto-frontal (FF) and FP EEG power spectrum. When administered in combination with citalopram, an attenuation of the power density reduction in the 7-15 Hz range in the FF EEG of citalopram alone, was observed. However, the EEG power spectral density and REM sleep suppressive effects of NAN-190 were both augmented. The results are compatible with the notion that serotonin is involved in the modulation of the slow wave activity in the EEG during NREM sleep. The results are cordant with other data suggesting that postsynaptic 5-HT1A stimulation might increase slow wave activity in the NREM EEG, and that serotonergic stimulation of other receptor subtypes (possibly 5-HT2) may decrease slow wave activity in the NREM EEG.     

Effect of α-helical-CRF[9-41] on feeding in goldfish: Involvement of cortisol and catecholamines.

The anoretic effect of corticotropin-releasing factor (CRF) was not dependent on adrenal activation in goldfish (Carassius auratus). Moreover, an interaction between CRF and the hypothalamic catecholaminergic system in the central regulation of food intake was observed. The intracerebroventricular (icv) administration of CRF increased cortisol levels and reduced food intake and hypothalamic norepinephrine and dopamine content at 2 hr postinjection, with these effects reversed by a-helical CRF[9–41] pretreatment. The anoretic effect of CRF was independent of the circulating cortisol increase, because it was only evoked after icv injections but not after intraperitoneal (ip) administration. Furthermore, the increase in plasma cortisol levels induced by ip administration of this steroid did not modify feeding. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Stimulation of neuropeptide Y overflow in the rat paraventricular nucleus by corticotropin-releasing factor

Neuropeptide Y (NPY) and corticotropin-releasing factor (CRF) are present at high concentrations in the hypothalamus where they mediate important endocrine and autonomic functions. Morphological and physiological studies have suggested an interaction between these peptides, and opposing actions of CRF and NPY have been reported on feeding and other behaviors. This study investigated the effect of CRF on NPY release in vivo, measured by push-pull techniques, in the anesthetized rat. Push-pull probes implanted into the paraventricular nucleus of the hypothalamus (PVN) were perfused with modified Ringer solution containing bovine serum albumin at 15 microl/min, and the perfusate was lyophilized prior to NPY radioimmunoassay. NPY overflow from the rat PVN was increased threefold by perfusion of a depolarizing concentration of potassium (50 mmol/L KCl). When CRF was administered into the PVN via the push-pull cannula at 1 or 5 microg/ml, dose-dependent increases in NPY overflow of two- and fivefold were observed (p < 0.05). These increases were abolished by prior intracerebroventricular (i.c.v.) administration of the CRF antagonist [D-Phe12,Nle(21,38),C(alpha)MeLeu32]CRF (12-41) at 1 or 5 microg/microl, respectively. NPY overflow returned promptly to resting levels following CRF administration. In contrast, when CRF was administered by i.c.v. bolus at a similar total dose (2 microg), no significant effect on NPY overflow was observed. These data provide in vivo evidence for an interaction between CRF and NPY at the level of the PVN.     

Effects of peripherally administered corticotropin-releasing factor (CRF) and a CRF antagonist: Does peripheral CRF activity mediate behavior of guinea pig pups during isolation?

Guinea pig pups vocalized and ambulated when first isolated in a test cage; at 1 and 24 hr, levels of these behaviors had waned, and pups frequently exhibited a crouched stance, eye-closing, and piloerection. Injection (SC) of corticotropin-releasing factor (CRF) prior to isolation diminished the initial vocalization and locomotor responses and induced pups to exhibit the crouched stance, eye-closing, and piloerection at the beginning of the isolation period. Pretreatment with a CRF-receptor antagonist reversed the behavioral effects of CRF. CRF had no effect on blood pressure. Thus, SC CRF produced the same behavioral profile as seen with the passage of time in untreated isolated pups. The behavioral effects appeared to be CRF-receptor-mediated events and were not secondary to hypotension. These results support the hypothesis that during prolonged isolation, high or sustained peripheral CRF activity modulates behavior. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Acute immobilization stress and intraventricular injection of CRF suppress naloxone-induced LH release in ovariectomized estrogen-primed rats

The present study was undertaken to evaluate the role and possible interaction of the endogenous opioid peptide (EOP) and corticotropin-releasing factor (CRF) in the acute stress-induced suppression of gonadotropin secretion in ovariectomized estrogen-primed rats. An intravenous (i.v.) injection of naloxone (10 or 20 mg/kg), an EOP antagonist, significantly elevated serum luteinizing hormone (LH) levels within 10 min in non-stressed animals. The naloxone-induced LH release was completely eliminated when tested 30 min after the onset of acute immobilization. In a subsequent study, it was found that suppression of the naloxone-induced LH release occurred as early as 5 min after the stress onset, and was still evident 60 min after the end of a 30-min period of immobilization. The effect of naloxone was restored 3 h after liberation of the animal from the 30-min immobilization. An intraventricular (i.c.v.) injection of CRF (1 or 5 micrograms) also significantly suppressed, in a dose-related manner, the effect of a subsequent i.v. injection of naloxone. However, an i.c.v. injection of alpha-helical CRF(9-41) (25 or 50 micrograms), a CRF antagonist, prior to immobilization, could not interfere with the suppressive effect of stress on naloxone-induced LH release. These results suggest that both acute immobilization stress and CRF can inhibit the LH secretory activity without mediation by EOP neurons. However, the stress-related suppression may involve non-CRF mechanism(s).     

The human neuroblastoma cell line, IMR-32, expresses functional corticotropin-releasing factor receptors

Corticotropin-releasing factor (CRF) receptors in IMR-32 human neuroblastoma cells were characterized after differentiation with 2.5 microM 5'-bromo-2'-deoxyuridine for 10 days. Scatchard analysis of [125I-Tyr0]ovine CRF binding revealed a high affinity binding site with a dissociation constant of 0.59 nM and a maximum binding capacity of 142 fmol/mg, the affinity of which was decreased by guanosine 5'-o-(3-thiotriphosphate). This binding was displaced in the following order of potency: human/rat CRF > ovine CRF > urotensin I > sauvagine > bovine CRF > [D-Phe12, Nle21,38, C alpha-MeLeu37]human/rat CRF-(12-41) > alpha-helical CRF-(9-41), indicative of the CRF1 receptor subtype. Functional coupling of this receptor was confirmed by CRF-induced increases in cyclic AMP, which were antagonised by alpha-helical CRF-(9-41) and [D-Phe12,Nle21,38,C alpha-MeLeu37] human/rat CRF-(12-41).     

Role of central interleukin-1 beta in gastrointestinal motor disturbances induced by lipopolysaccharide in sheep

Cytokines are involved in the symptoms of the acute phase response induced by infectious diseases in humans as well as in animals, and interleukin-1 beta (IL-1 beta) has a pivotal role in these changes. The role of central IL-1 beta in the gastrointestinal hypomotility and fever evoked by intravenous administration of lipopolysaccharide (LPS) and the mechanisms involved, were investigated in sheep as an experimental model. LPS (0.1 microgram/kg, intravenously) induced gastrointestinal hypomotility and fever that were significantly reduced by prior intracerebroventricular administration of IL-1 receptor antagonist protein (IL-1ra, 2 micrograms/kg). The effects of LPS were mimicked by intracerebroventricular IL-1 beta (50 ng/kg), whereas IL-1 beta injected intravenously at the same dose only caused a slight and transient fever without modifying the gastrointestinal motility. Prior intracerebroventricular administration of the cyclooxygenase inhibitor indomethacin (100 micrograms/kg) but not the corticotropin-releasing factor (CRF) receptor antagonist alpha-helical CRF9-41 (5 micrograms/kg) blocked all effects by both LPS and IL-1 beta. These results suggest that in sheep, LPS induces digestive motor disturbances through a central release of IL-1 beta and prostaglandins.     

Dynamics of the effects of levoprotiline and maprotiline on EEG in patients with major depressive episodes (DSM-III-R)

The presented study compares the effect of the well-tested antidepressant maprotiline and a new antidepressant with an atypical pharmacological profile, levoprotiline, on EEG during repeated assessment after single dose administration. From the original number of 34 patients fulfilling the criteria of a major depressive episode (DSM-III-R) on account of a low-voltage record or pathological findings 11 were eliminated. To 12 of the remaining patients levoprotiline was administered and to 11 maprotiline, after a one-week placebo period, in doses of 150 mg. The EEG was recorded after an accommodation session immediately before, 1.5, 3, 4.5, 6 and 24 hours after a single dose administration. The record was taken on a 16-channel average reference montage at rest with closed eyes. Two-minute intervals were divided into 30 four-second periods at a sampling frequency of 128 Hz. From the signal by means of FFT the spectra were estimated and the mean spectrum for the entire recording was calculated. This was then divided into 10 frequency bands. The new method of frequency analysis of alpha-entropy was also used which is a global measure of the difference between two spectra. Three hours after administration of a single dose levoprotiline had an EEG profile corresponding to the profile of tricyclic antidepressants, i.e. it increased the values of the power spectra density in the region of 5-8.5 Hz and in the entire beta band; the decline of power in the alpha band was, however, absent. As regards maprotiline, 3 hours after administration a profile typical for antidepressants was not found; obviously because of the great variance of values of power spectra density as a result of great interindividual differences in the ingestion phase. Changes of the EEG spectrum expressed as values of alpha-entropy during different periods of apparently assessment are not incidental. After the initial rise of values a decline occurs.     

Novel clinical approaches in monoclonal antibody-based management in colorectal cancer patients: radioimmunoguided surgery and antigen augmentation

In animals, changes in brain temperature induce a shift in frequencies in the electroencephalogram (EEG). Given the large decreases in body and brain temperature that occur during hibernation, putative functions that were previously ascribed to certain EEG frequencies are no longer valid because of the progressive shift away from the original frequency. In the present review it is proposed that even moderate temperature changes in humans and animals, such as those across the circadian or menstrual cycle, or induced by drugs, have a significant effect on EEG frequencies and the corresponding power spectrum. Alterations in the relative EEG power spectrum, in studies where body temperature also changes, may not be a direct cause of the treatment under investigation, but a consequence of effects on body or brain temperature. However, these effects on the EEG power spectrum are usually interpreted to result directly from the experimental treatment.     

Advances and controversies in the diagnosis and management of medullary thyroid carcinoma

In tasks requiring sustained attention, human alertness varies on a minute time scale. This can have serious consequences in occupations ranging from air traffic control to monitoring of nuclear power plants. Changes in the electroencephalographic (EEG) power spectrum accompany these fluctuations in the level of alertness, as assessed by measuring simultaneous changes in EEG and performance on an auditory monitoring task. By combining power spectrum estimation, principal component analysis and artificial neural networks, we show that continuous, accurate, noninvasive, and near real-time estimation of an operator's global level of alertness is feasible using EEG measures recorded from as few as two central scalp sites. This demonstration could lead to a practical system for noninvasive monitoring of the cognitive state of human operators in attention-critical settings.     

Effects of leptin on corticotropin-releasing factor (CRF) synthesis and CRF neuron activation in the paraventricular hypothalamic nucleus of obese (ob/ob) mice

The effects of leptin on the levels of CRF messenger RNA (mRNA) in the paraventricular hypothalamic nucleus (PVN), on the activation of the PVN CRF cells, and on the plasma levels of corticosterone were investigated in lean (+/?) and obese (ob/ob) C57BL/6J male mice. Murine leptin was s.c. infused using osmotic minipumps. The treatment period extended to 7 days, and the daily dose of leptin delivered was 100 microg/kg. The mice were killed either in a fed state or following 24 h of total food deprivation. The starvation paradigm was employed to enhance the activity of the hypothalamic-pituitary-adrenal axis in obese mice. In situ hybridization histochemistry was performed to determine the PVN levels of CRF mRNA and the arcuate nucleus levels of neuropeptide Y mRNA. The activity of the PVN CRF cells was estimated from the number of PVN cells colocalizing CRF mRNA and the protein Fos. Leptin led to a reduction in body weight gain and fat deposition. These effects were seen in both +/? and ob/ob mice and were observed to be particularly striking in obese mutants, in which leptin also caused an important reduction in food intake. Leptin also was found to affect plasma levels of corticosterone. It lowered the high corticosterone levels of obese mutants, an effect that appeared more evident in food-deprived than in fed mice. Finally, leptin prevented the induction of CRF synthesis in the PVN and the activation of the PVN CRF neurons observed in food-deprived ob/ob mice and hindered the elevation of arcuate nucleus neuropeptide Y synthesis in ob/ob mice. Together these results suggest a role for leptin in the excessive response of the hypophysiotropic CRF system of the ob/ob mouse.     

Relaxing actions of corticotropin-releasing factor on rat resistance arteries

1. Although it well established that corticotropin-releasing factor (CRF) injected i.v. can cause hypotension and vasodilatation, there is no in vitro evidence that CRF acts as a vasodilator. We have therefore tested the hypothesis that the hypotensive effect of i.v. CRF is due to a direct vasodilator action by carrying out experiments in vitro on rat resistance arteries (i.d. 150-300 microns). 2. Initial in vivo experiments confirmed that CRF (1.5 nmol.kg-1) injected i.v. caused hypotension in rats, this being partially antagonized by the CRF analogue CRF9-41. 3. For the in vitro experiments, vessels were taken from the mesenteric, cerebral and femoral vascular beds, and mounted as ring preparations in an isometric myograph. The vessels were pre-contracted with one of 3 agonists (prostaglandin F2 alpha, arginine vasopressin or noradrenaline) or with a high-potassium solution (K+). 4. With maximal concentrations of the agonists, CRF caused relaxation of mesenteric and cerebral vessels with 10 nM, and near complete relaxation with 100 nM. Femoral vessels pre-constricted with agonists and all vessels pre-constricted with K+ were less affected by CRF. In the mesenteric vessels, with sub-maximal levels of pre-constriction, CRF caused substantial relaxation at 1 nM and could cause complete relaxation at 10 nM. 5. The relaxant effect of CRF on contractions of mesenteric vessels was antagonized by 100 nM CRF9-41. Neither tetraethyl ammonium (30 mM) nor glibenclamide (3 microM) antagonized the relaxant effect of CRF. 6. The relaxant effect of CRF on mesenteric small arteries was found to be unaffected by removal of the endothelium. 7. The results indicate that CRF causes an endothelial-independent vasodilatation of rat resistance arteries under in vitro conditions at concentrations which are consistent with this being an important cause of the hypotension observed with i.v. injection of CRF.     

Distal lateral ventricular atrium: reevaluation of normal range

This study investigated the involvement of corticotrophin-releasing factor (CRF) in acute neuronal damage induced by focal cerebral ischaemia or pharmacological activation of NMDA receptors in the rat brain. Intracerebroventricular injection of a CRF receptor antagonist (alpha-helical CRF9-41), markedly inhibited ischaemic (61%) and excitotoxic (41%) brain damage. Peripheral injection of a glucocorticoid antagonist (RU38486) did not affect ischaemic damage. Ischaemic and excitotoxic damage caused increased hypothalamic concentrations of CRF. These data indicate that CRF mediates ischaemic and excitotoxic neuronal damage in the rat, but that this effect is not dependent on glucocorticoids.     

CRF type I receptor-deficient mice exhibit a pronounced pituitary-adrenal response to local inflammation

Recent studies indicate that the regulation of adrenocorticotropin (ACTH) secretion by corticotropin-releasing factor (CRF) is mediated predominantly by the type I CRF receptor (CRF-R1). Indeed, CRF-R1-deficient (CRF-R1 -/-) mice show marked impairment of the pituitary-adrenal axis. However, the plasma ACTH concentrations of unstressed CRF-R1 -/- mice are similar to those in wild-type mice. We show here that arginine vasopressin (AVP) is a major ACTH secretagogue in CRF-R1 -/- mice in resting conditions, since administration of anti-AVP serum, but not anti-CRF serum, markedly reduced (by 60%) resting plasma ACTH concentrations in these mutants. We also investigated the pituitary-adrenal response to turpentine-induced local inflammation in CRF-R1 -/- mice. Administration of turpentine into the hind-limb of CRF-R1 -/- mice produced a slightly (15-25%) smaller swelling of the limb, but a 10 fold greater rise in plasma IL-6 levels, compared to CRF-R1 +/+ controls. Turpentine-induced local inflammation produced pronounced elevations in the plasma concentrations of both ACTH and corticosterone in both CRF-R1 -/- and wild-type mice, but ACTH secretion could be inhibited by anti-CRF and anti-AVP sera only in wild-type mice. These data indicate that resting ACTH secretion in CRF-R1 -/- mice is in part attributable to AVP-dependent mechanisms. Furthermore, while in normal mice the pituitary-adrenal response to local inflammation is mediated largely via CRF-dependent mechanisms, mice deficient in CRF-R1 are still able to mount a pituitary-adrenal response via mechanisms that do not depend critically on either CRF or AVP action.