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Degarelix is a potent very long-acting GnRH antagonist after subcutaneous administration. In this paper, we describe the synthesis of two analogs of degarelix incorporating racemic 3-(2-methoxy-5-pyridyl)-alanine (2-OMe-5Pal, 5) at position 3. The two diastereomers were separated by reverse-phase high-performance liquid chromatography (RP-HPLC) and the absolute stereochemistry at position 3 in the peptides was determined by enzymatic digestion with proteinase K. These analogs were tested in vitro for their ability to antagonize the GnRH receptor and in vivo for duration of action in a castrated male rat assay. Analog 7 with D2-OMe-5Pal was potent in vitro (IC50 = 5.22 nM); however, analog 8 with L2-OMe-5Pal at position 3 in degarelix lost potency as an antagonist of the human GnRH receptor (IC50 = 36.95 nM). Both the analogs were found to be short-acting in vivo.

In three earlier papers, the structures and biological potencies of numerous mono- and dicyclic antagonists of GnRH were reported. Among these, two families, each containing two to four members were identified that had very high antagonist potencies in an antiovulatory assay (within a factor of 2 of those of the most potent linear analogues) and high affinities (K(i)0.5 nM) for the rat GnRH receptor (rGnRHR). The most favored cycles bridged the side chains of residues (4-10),(1,2) (5-8),(2) (4-10/5-8),(2) (1-3),(3) and (1-3/4-10).(3) Our goal was to identify a consensus model of bioactive conformations of GnRH antagonists, yet these biocompatible constraints did not sufficiently restrain the spatial location of the N-terminal tripeptide with respect to the C-terminal heptapeptide, due largely to the rotational freedom about the bonds connecting these regions. Examination of models derived from NMR studies of cyclo(4-10) analogues suggested a large number of possible cyclic constraints such as cyclo (0-8), (1-8), or (2-8). All analogues tested with these substitutions were inactive as antiovulatory agents at 1 mg/rat (5-9) and had low affinity for rGnRHR. On the other hand, bridging positions 3 and 8 with a [DAsp(3)] to [Dbu(8)] (12, K(i) = 13 nM) or [Orn(8)] (13, K(i) = 14 nM) in the parent compound cyclo(3-8)[Ac-DNal(1),DCpa(2),DXaa(3), Arg(5),DNal(6),Xbb(8),DAla(10)]GnRH yielded analogues that blocked ovulation at 250 microgram/rat. Analogue 14 (K(i) = 2.3 nM), with a [DAsp(3), Lys(8)] bridge, was fully active at 50 microgram/rat. Loss of potency (20-fold) was observed with the substitution of [DAsp(3)] in 14 by [DGlu(3)] in 15 (K(i) = 23 nM). Dicyclic analogues possessing the (4-10) cycle and selected (1-6), (2-6), and (2-8) cycles led to analogues that were inactive at doses of 500 microgram/rat or larger. Two analogues with (1-8/4-10) cycles (16, K(i) = 1.1 nM) or (3-8/4-10) cycles (22, K(i) = 17 nM) showed full antiovulatory potency at 250 microgram/rat. None of these substitutions yielded analogues potent enough (80% inhibition of ovulation at 5 microgram/rat or less and K(i)0.5 nM) to be candidates for structural analysis by NMR. On the other hand, four dicyclic (1, 1'-5/4-10) analogues met this criterion: dicyclo(1, 1'-5/4-10)[Ac-Asp(1)(Gly),DCpa(2),DTrp(3),Asp(4),Dbu(5 ), DNal(6), Dpr(10)]GnRH (32, K(i) = 0.22 nM), dicyclo(1, 1'-5/4-10)[Ac-Asp(1)(Gly),DCpa(2),DNal(3),Asp(4),Dbu(5 ), DNal(6), Dpr(10)]GnRH (34, K(i) = 0.38 nM), dicyclo(1, 1'-5/4-10)[Ac-Asp(1)(betaAla),DCpa(2), DTrp(3),Asp(4),Dbu(5),DNal(6), Dpr(10)]GnRH (40, K(i) = 0.15 nM), and dicyclo(1, 1'-5/4-10)[Ac-Glu(1)(Gly), DCpa(2),DTrp(3),Asp(4),Dbu(5),DNal(6), Dpr(10)]GnRH (41, K(i) = 0.24 nM). Since they differed slightly in terms of the (1,1'-5) bridge length (21 and 22 atoms) and bridgehead configuration, we may hypothesize that they assume similar bioactive conformations that satisfy a very discriminating receptor, since many other closely related analogues were significantly less potent.

The characteristics of a high-affinity antagonist radioligand are compared with those a high-affinity agonist in binding to the cloned corticotropin-releasing factor receptor type 1 (CRF-R1) and type 2 (CRF-R2) and to the native receptors that exist in rat cerebellum and brain stem. The relative potencies of CRF antagonists and agonists to the two types of cloned CRF receptors overexpressed stably in Chinese hamster ovary cells are determined using the antagonist radioligand 125I- [DTyr1]astressin (Ast*), and the agonist radioligand, 125I -[Tyr0]rat urocortin (Ucn*). The inhibitory binding constants (Ki) of astressin and urocortin are 1 to 2 nM for all receptors and are independent of which radioligand is employed. Astressin binds with high affinity to the native cerebellar/brain stem receptor and relative potencies of selected CRF analogs determined with Ast* on the native receptor are similar to those obtained for the cloned CRF-R1. The specific binding of Ast* to endogenous brain receptors is greater than that of Ucn*, resulting in more sites being detected by the antagonist than by the agonist. In contrast to another CRF agonist, the binding of Ucn* to the cloned receptors is relatively insensitive to guanyl nucleotides at both 20 degreesC and 37 degreesC; however, its binding to the native receptor is displaced by guanyl nucleotides at 37 degreesC and, to a lesser degree, at 20 degreesC. As expected, the binding of the antagonist Ast* is not affected by guanyl nucleotides. Because it is a high-affinity, specific CRF antagonist, astressin is eminently suitable as a ligand for detection and characterization of both endogenous and cloned CRF receptors.

Urocortin (Ucn) is a newly identified mammalian member of the CRF family of peptides. Ucn activates CRF receptors (both CRF-R1 and CRF-R2) with greater potency than CRF itself, suggesting that Ucn may play an endogenous role in eliciting (at least some) CRF receptor-mediated events. Because the most characterized physiological function of CRF receptors is the activation of pituitary ACTH secretion, we have compared the effects and potential endogenous roles of CRF and Ucn in regulating plasma ACTH concentrations in intact male rats. Synthetic rat Ucn injected i.v. (0.09-9.0 nmol/kg) elicited ACTH secretion in a dose-dependent manner, causing greater ACTH secretion than CRF at each dose tested. The increases in plasma ACTH concentrations produced by CRF or Ucn were virtually abolished by pretreatment with the CRF receptor antagonist, astressin (3 mg/kg), and were partially attenuated (by 27-37%) by an antiarginine vasopressin serum. These data indicate that both Ucn and CRF elicit ACTH secretion via CRF receptor-dependent mechanisms, and that the ACTH-releasing activities of both CRF and Ucn are potentiated by endogenous arginine vasopressin. Intravenous administration of rabbit anti-Ucn serum, which inhibited ACTH secretion produced by Ucn, but not CRF, had no statistically significant effect on either resting (midday) plasma ACTH concentrations or the rise in ACTH levels elicited by 30 min of intermittent electrofootshocks. By contrast, treatment with a rabbit anti-CRF serum that specifically inhibited the ACTH response to CRF lowered plasma concentrations in control unstressed rats and largely prevented the plasma ACTH response to electrofootshocks. These data indicate that although Ucn is a more potent ACTH secretagogue than CRF in the intact male rat, it is not a major endogenous regulator of pituitary ACTH secretion under basal (midday) conditions or during acute footshock stress.

Neuropeptide Y (NPY) has neuromodulatory actions on multiple brain functions including endocrine, behavioral, and circadian processes and has been implicated in the pathophysiology of both anxiety and depression. Behavioral studies suggest that NPY is a potent anxiolytic, whereas CRF is anxiogenic, thus it seems that a balance of these two peptides may exert important influences on behavioral state regulation. However, little is known about how the NPY/CRF balance affects general arousal, attention, and/or sleep states. The present study evaluated the effects of CRF alone, and co-administered with NPY, on spontaneous brain activity as well as on auditory processing using electrophysiological measures. Electroencephalographic (EEG) and event-related potentials (ERPs) were obtained in rats following intracerebroventricular administration of CRF (0.5 microgram) and CRF (0.5 microgram)/NPY (5.0 or 15 micrograms). Auditory processing, as assessed by ERPs, was affected most significantly in the frontal cortex where CRF produced increases in the N1 and P3 components of the ERP, and NPY/CRF co-administration produced significant decreases. These data are consistent with a role for CRF in hyperarousal, and further suggest that NPY may be capable of reversing such states. Administration of CRF also produced a significant increase in the time to sleep onset and a decrease in the amount of time spent in non-rapid eye movement (NREM) sleep as quantified by scoring the EEG paper records. Co-administration of NPY with CRF reversed the effects of CRF on sleep duration and sleep onset in a dose-dependent fashion. Spectral analysis revealed that CRF produced quantitative changes in the EEG that were similar to what has previously been reported. CRF-induced increases in fast frequency activity were found to be reversed by co-administration of NPY. Taken together these data suggest that "dysregulation" of sleep and arousal states in depression and anxiety may be consistent with an upset of the balance between hypothalamic neuropeptide systems.

We report the isolation, characterization, and total synthesis of a small peptide ligand for nicotinic acetylcholine receptors (nAChRs). It is highly active against the neuromuscular receptor in frog but not in mice. In contrast, it induces seizures when injected centrally in mice and rats, suggesting that it may target neuronal nAChRs in mammals. Although such receptors may be important in both normal cognition and the pathophysiology of several neuropsychiatric disorders, there are few ligands to discriminate between the multiple receptor subtypes. The new peptide is a highly divergent alpha-conotoxin from the snail Conus imperialis, which preys on polychaete worms. In this article, the purification, structural analysis, synthesis, and preliminary physiological characterization of alpha-conotoxin ImI (alpha-CTx-ImI) are reported. The sequence of the peptide is: Gly-Cys-Cys-Ser-Asp-Pro-Arg-Cys-Ala-Trp-Arg-Cys-NH2. The peptide shows striking sequence differences from all alpha-conotoxins of fish-hunting Conus, but its disulfide-bridging is similar: [2-8; 3-12]. We suggest that cone venoms may provide an array of ligands with selectivity for various neuronal nAChR subtypes.

Neuropeptide Y (NPY) is a 36-amino-acid, C-terminal amidated peptide that is found in bulbospinal pathways and can inhibit the release of the primary afferent C-fiber neurotransmitter, substance P. Based on these observations, the present studies examined the possible antinociceptive effects of this peptide and several NPY fragments after intrathecal administration in rats prepared with chronic intrathecal catheters. In the 52 degrees C hot plate test, NPY produced a dose-dependent elevation in the nociceptive threshold with a median effective dose of 1.1 nmol. The ordering of fragments' activity was: NPY greater than NPY16-36 greater than or equal to NPY19-36 greater than or equal to NPY14-36 greater than or equal to NPY18-36 much greater than NPY1-36-OH = NPY18-36-OH = 0. In the paw pressure test, NPY was not active, even at the highest doses examined (median effective dose greater than 20 nmol), whereas the C-terminal fragments retained their potency and produced significant increases in the pressure required to evoke escape (NPY18-36: median effective dose = 18.7 nmol). The rank ordering of activity in the paw pressure test was: NPY19-36 greater than or equal to NPY14-36 greater than or equal to NPY18-36 greater than or equal to NPY16-36 much greater than NPY = NPY18-36-OH = 0. Peptide YY, human pancreatic polypeptide and avian pancreatic polypeptide behave similarly to NPY.(ABSTRACT TRUNCATED AT 250 WORDS)

We studied the possibility of multiple neuropeptide Y (NPY) receptor subtypes. NPY-stimulated Ca2+ mobilization in human erythroleukemia (HEL) cells was used to screen a number of NPY analogues. The potencies of three of these analogues [peptide YY (PYY), [D-Tyr-36]NPY, and NPY-(18-36)] were compared with that of NPY in the following model systems: Ca2+ mobilization and inhibition of adenosine 3',5'-cyclic monophosphate accumulation in HEL cells, potentiation of vasoconstriction in the isolated rabbit ear artery, reduction of cutaneous microvascular perfusion in the rat digit, and inhibition of [3H]serotonin release in rat brain. In each of the five models, PYY was a full agonist that exhibited a similar or slightly higher potency than NPY, whereas [D-Tyr-36]NPY and NPY-(18-36) were partial agonists with lower potencies: NPY-(18-36) had a lower potency and efficacy than [D-Tyr-36]NPY in HEL cells and the rabbit ear artery, but was more effective than [D-Tyr-36]NPY for constricting cutaneous microvasculature and inhibiting serotonin release. Because of its weak partial agonism, we also tested NPY-(18-36) as an antagonist of NPY-stimulated Ca2+ mobilization in HEL cells. NPY-(18-36) shifted the NPY concentration-response curve to the right with a KB affinity value of 297 nM. In summary, [D-Tyr-36]NPY and NPY-(18-36) are partial agonists, the relative potency of which varies between systems. These data demonstrate the presence of multiple NPY receptor subtypes. We propose a modified classification scheme of NPY receptor subtypes.

We have studied the hemodynamic effects of neuropeptide Y (NPY) and its COOH-terminal fragment NPY-(18–36) in conscious rats. Intra-arterial injection of NPY rapidly elevated systemic vascular resistance (SVR), which remained high for greater than 30 min. Cardiac output (CO) decreased, and it remained low for greater than 30 min. Accordingly, blood pressure rose only transiently and returned to base-line values within 5 min. The reduction of CO could be attributed to a decreased stroke volume with an only marginal reduction of heart rate. Thus a direct cardiodepressive effect of NPY rather than baroreflex activation appears to be the major cause of the reduced CO. In vitro experiments excluded the possibility that NPY has direct negative inotropic effects and suggest that its cardiodepressive action is caused by coronary vasoconstriction or by presynaptic inhibition of norepinephrine release. Intra-arterial injections of NPY-(18-36) caused different hemodynamic effects. NPY-(18–36) decreased CO in a manner similar to that seen with NPY but initially did not elevate SVR, resulting overall in a reduced blood pressure. Only later, when blood pressure was reduced, was an elevation of SVR observed, which could be associated with increased plasma levels of catecholamines, angiotensin II, vasopressin, and NPY. Thus NPY-(18–36) mimics the cardiac effects of NPY but does not elicit its vascular effects. As NPY-(18–36) discriminates between NPY receptor subtypes in vitro, we conclude that the cardiac and vascular effects of NPY are mediated by distinct receptor subtypes.

The central nervous system effects of thyrotropin-releasing hormone (TRH) on proximal duodenal bicarbonate secretion were studied in freely moving rats. Cerebroventricular administration of TRH (0.5-5.0 nmol) significantly stimulated basal duodenal bicarbonate secretion, whereas intravenous administration of TRH did not. Ganglionic blockade with chlorisondamine and truncal vagotomy abolished TRH-induced bicarbonate secretion, whereas atropine significantly attenuated the response. The vasoactive intestinal peptide (VIP) receptor antagonist, (4Cl-D-Phe6, Leu17) VIP given intravenously completely prevented the stimulatory effect of central TRH on duodenal bicarbonate secretion. In contrast, hypophysectomy, adrenalectomy, opiate and noradrenergic blockade, or indomethacin did not affect the TRH-induced bicarbonate response. Intravenous administration of VIP and carbachol significantly stimulated bicarbonate outputs, and these responses were blocked by the VIP antagonist and atropine, respectively. These results indicate that TRH may serve as a central nervous system mediator that stimulates duodenal bicarbonate secretion in rats by increasing vagal outflow. Vagal stimulation induced by TRH increases duodenal bicarbonate secretion by the release of VIP and, in part, by activation of a muscarinic pathway but not by pituitary, adrenal, and noradrenergic pathways or endogenous opiates and prostaglandins. The actions of peripheral VIP and carbachol appear to be mediated by specific VIP and muscarinic receptors, respectively.

Systematic substitution of the natural L-amino acids in neurotensin by their D isomers reveals that the COOH-terminal portion of this tridecapeptide is required for binding to mast cell receptors: D-amino acid replacements from Pro10 through Leu13 substantially decrease that binding. Either blockage of the COOH-terminal carboxyl group as with N-methylamidation, or formation of a cyclic structure by the inclusion of a disulfide bond, a Cys2,13 substitution, markedly reduces the specific binding to mast cell receptor sites. Modifications in the NH2-terminal portion of neurotensin do not affect the binding to mast cells. However, D-Arg8 and D-Arg9 substitutions increase binding by factors of 5- to 6-fold. The hydroxyl group at position 3 or 11 is not essential for binding since Phe3 or Phe11 is equivalent to Tyr3 or Tyr11. The COOH-terminal penta- and hexapeptides are able to displace approximately 70% 125I-neurotensin relative to the intact peptide. Of 18 other biologically active peptides tested, only xenopsin, a naturally occurring COOH-terminal analog of neurotensin, and bradykinin effectively compete in the binding assay to an extent of 60 and 100%, respectively. Histamine, diphenhydramine, and noradrenaline are ineffective in this regard.