"Selective" D-1 and D-2 receptor antagonists fail to differentially alter supersensitive locomotor behavior in the rat

The dopamine receptor antagonists SCH 23390 and spiperone show highly selective in vitro affinity for D-1 and D-2 dopamine receptor subtypes, respectively. We studied the effects of these selective antagonists on the supersensitive locomotor response to apomorphine in rats following 6- hydroxydopamine (6OHDA) lesions of the nucleus accumbens (N. Acc.). Both D-1 and D-2 receptor antagonists produced dose-dependent blockade of the supersensitive locomotor response at doses that did not depress baseline locomotor activity. The behavioral properties of these D-1 and D-2 receptor antagonists were further examined using a simple step-down motor task. Both antagonists produced catalepsy as evidenced by dose-dependent increases in step- down latency. These results indicate that drugs with distinct in vitro dopamine binding affinities cannot be distinguished on the basis of their ability to inhibit supersensitive locomotor activity or simple motor tasks in rats in vivo.     

Synthesis and structure-activity relationship of RXR antagonists based on the diazepinylbenzoic acid structure

Synthesis and structure-activity relationship of RXR antagonists employing a diazepinylbenzoic acid scaffold are described. Of those antagonists, sulfonamide derivatives (6v and 6w) reveal a high antagonistic activity and good pharmacokinetic properties.     

Increased calcitonin gene-related peptide in neuroma and invading macrophages is involved in the up-regulation of interleukin-6 and thermal hyperalgesia in a rat model of mononeuropathy

The pain related peptide, calcitonin gene-related peptide (CGRP), plays an important role in inflammatory pain and immune responses. However, its role in neuropathic pain is not established. Following nerve injury, CGRP and pro-inflammatory interleukin-6 (IL-6) are increased in injured nerves. The aim of this study was to determine if CGRP in injured nerves is involved in the up-regulation of IL-6 and in the maintenance of neuropathic pain. Perineural injection of a neutralizing IL-6 antiserum or CGRP receptor antagonists (CGRP8-37 and BIBN4096BS) effectively attenuated thermal hyperalgesia 4 weeks after partial sciatic nerve ligation. Perineural CGRP antagonists also dramatically reduced IL-6 level in injured nerves. CGRP release from injured sites was dramatically increased and CGRP immunoreactivity was localized in both neuroma and invading macrophages. CGRP receptor markers (CRLR and RAMP1) were expressed in invading macrophages. Both CGRP antagonists significantly reduced IL-6 release from injured nerve explants. In cell cultures derived from injured nerves, CGRP concentration-dependently increased IL-6 release, an effect also blocked by CGRP antagonists. Taken together, these data show that increased levels of CGRP in injured neuroma and invading macrophages are involved in the up-regulation of IL-6 in macrophages as well as in the maintenance of neuropathic pain.     

Inverse agonists and neutral antagonists at µ opioid receptor (MOR): possible role of basal receptor signaling in narcotic dependence

The mu opioid receptor, MOR, displays spontaneous agonist-independent (basal) G protein coupling in vitro. To determine whether basal MOR signaling contributes to narcotic dependence, antagonists were tested for intrinsic effects on basal MOR signaling in vitro and in vivo, before and after morphine pretreatment. Intrinsic effects of MOR ligands were tested by measuring GTPgammaS binding to cell membranes and cAMP levels in intact cells. beta-CNA, C-CAM, BNTX, and nalmefene were identified as inverse agonists (suppressing basal MOR signaling). Naloxone and naltrexone were neutral antagonists (not affecting basal signaling) in untreated cells, whereas inverse agonistic effects became apparent only after morphine pretreatment. In contrast, 6alpha- and 6beta-naltrexol and -naloxol, and 6beta-naltrexamine were neutral antagonists regardless of morphine pretreatment. In an acute and chronic mouse model of morphine-induced dependence, 6beta-naltrexol caused significantly reduced withdrawal jumping compared to naloxone and naltrexone, at doses effective in blocking morphine antinociception. This supports the hypothesis that naloxone-induced withdrawal symptoms result at least in part from suppression of basal signaling activity of MOR in morphine-dependent animals. Neutral antagonists have promise in treatment of narcotic addiction.     

A novel synthesis of 2-arylpyrrolo[1,2-a]pyrimid-7-ones and their structure-activity relationships as potent GnRH receptor antagonists

In the process of developing GnRH receptor antagonists, a novel base-catalyzed cyclization of compounds 5a-b was discovered, which led to the formation of the 2-aryl pyrrolo[1,2-a]pyrimid-7-one core structures 6a-b. These intermediates were further modified at positions 1, 2, 4 and 6 to afford a series of potent GnRH antagonists with low nanomolar K(i) values.     

Receptor-mediated uptake of GnRH agonist and antagonists by cultured gonadotropes: evidence for differential intracellular routing

The binding and uptake of the GnRH agonist D-Lys6-GnRH and of the antagonists [N-Ac-D-(pyro)-Cl-Phe1,2-D-Trp3-Lys6-D-Ala10]-GnRH and D-p-Glu1-D-Phe2-D-Trp3-D-Lys6-GnRH by dispersed pituitary gonadotropes was studied with electron microscopy. The peptides were coupled to colloidal gold markers with a diameter of 6 or 20 nm which were incubated separately or together for time periods between 15 and 180 min. Both antagonists could be found after 45 and 180 min at 37 degrees C in lysosomes as well as at the plasma membrane of gonadotropes. Co-incubation of both antagonists or of agonist and either antagonist resulted in uptake of the conjugates into separate lysosomes as well as mixed together into the same lysosome. Localization of the antagonists in structures associated with the Golgi apparatus was not observed at the time points studied. The results show that both GnRH agonist- and antagonist-conjugates are biologically active and that they are internalized by the gonadotropes via receptor mediated endocytosis. The failure to detect antagonist conjugates in the Golgi apparatus may indicate that passage through this organelle requires activation of the receptors by agonists and that the uptake of antagonist into lysosomes due to normal membrane protein turnover.     

The effects of several ion channel blockers and calmodulin antagonists on fertilization-induced acid release and 45Ca2+ uptake in sea urchin eggs

The effects of calcium antagonists, diltiazem and verapamil, and calmodulin antagonists, chlorpromazine, N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), were tested on two responses of the sea urchin egg to insemination: (1) H+ release; (2) Ca2+ uptake. It was found that calcium antagonists inhibited both processes, while calmodulin antagonists only inhibited H+ release but not Ca2+ uptake. Verapamil and diltiazem were effective to inhibit H+ release when added to the egg suspension up to 120 sec and W-7 was effective around 150 sec after insemination. Calcium antagonists became ineffective earlier than W-7 in inhibiting H+ release. A calmodulin-dependent step may thus occur linking the Ca2+ uptake and H+ release. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an anion channel blocker, also inhibited both Ca2+ uptake and H+ release. This result suggests that an uptake of anion(s) occurs along with Ca2+ uptake.     

Glucose transport inhibition in human erythrocytes by calmodulin antagonists

Calmodulin antagonists (tryphtazin, lidocaine, dykain, palmitate) inhibit glucose transport from human erythrocytes. Glucose efflux inhibition is proportional to the concentration of antagonists in the medium and is of uncompetitive character. It is accompanied by a decrease in the maximum transport rate with the unchanged constant of dissociation in the complex: carrier-sugar. Calcium ionophores A23187 and divaleryldibenzo-18-crown-6 eliminated the inhibiting effect of pharmacological agents on glucose transport. The authors think that the glucose transport inhibition under the influence of calmodulin antagonists may be realized through the calmodulin-dependent chain inhibition under the influence of calmodulin antagonists in the carbohydrate transport system.     

Pharmacological characterization of dopamine receptors in the corpus allatum of Manduca sexta larvae

Dopamine receptors previously identified in corpora allata (CA) of Manduca sexta last instars on the basis of dopamine effects on JH (juvenile hormone)/JH acid biosynthesis and cyclic AMP (cAMP) accumulation, were characterized pharmacologically. For this study, a broad spectrum of agonists or antagonists of D1, D2, D3 or D4 dopamine receptors, together with the dopamine metabolite N-acetyl-dopamine, other neurotransmitters and their agonists/antagonists, were tested for their effects on gland activity and cAMP production. The lack of effect of other neurotransmitters supports the specificity of the effect of dopamine and the dopamine specificity of the receptors. Only the D2 receptor antagonist spiperone had a potent effect on JH biosynthesis and cAMP formation by CA taken on day 0 of the last stadium, when dopamine stimulates both activities and thus appears to be acting via a D1-like receptor. Several other D2 receptor antagonists, and D1, D2/D1 and D4,3/D2 receptor antagonists were less effective. Thus, the D1-like receptor of the Manduca CA appears to be distinct pharmacologically from vertebrate D1 receptors. By contrast, a number of D2 agonists/antagonists had a significant effect on JH acid biosynthesis and cAMP production by the CA from day 6 of the last stadium, when dopamine inhibits both activities and thus appears to be acting via a D2-like receptor. Certain D1-specific agonists/antagonists were equally effective. The Manduca D2-like receptor therefore bears some pharmacological resemblance to vertebrate D2 receptors. N-acetyl dopamine acted as a dopamine agonist with day 6 CA, the first identified function for an N-acetylated biogenic amine in insects. Dopamine was found to have the same differential affect on the formation of cAMP in homogenates of day 0 and day 6 brains as it did with CA, and in the same concentration range. Dopamine receptor agonists/antagonists affecting cAMP formation by day 0 and day 6 CA homogenates had similar effects with brain homogenates. By contrast, dopamine only stimulated cAMP formation by homogenates of day 0 and day 6 abdominal or ventral nerve cord. These results suggest that D1- and D2-like dopamine receptors of Manduca are regionally as well as temporally localized.     

Inhibition of in vivo and in vitro fertilization in rodents by gonadotropin-releasing hormone antagonists

We have examined the effect of two GnRH antagonists, Ac-D-Nal(1)-Cl-D-Phe(2)-3-Pyr-D-Ala(3)-Arg(5)-D-Glu(AA)(6)-GnRH (Nal-Glu) and Ac(3,4)-dehydro-Pro(1),-p-fluoro-D-Phe(2),D-Trp(3,6)-GnRH (4pF), on in vivo and in vitro fertilization in rodents. Female rats were treated in the afternoon of proestrus with 2 micro l of Nal-Glu or 4pF (0.5 and 5 mM) injected directly into one oviductal horn (experimental); saline was injected into the contralateral horn (control). Females were then mated and the oviducts were perfused for egg and sperm recovery. The results indicate that both antagonists inhibited in vivo fertilization. Thus, the percentage of fertilized eggs in control oviducts ranged from 92% +/- 5% to 100% +/- 0%, whereas in treated oviducts, fertilization ranged from 25% +/- 6% to 73% +/- 5%. GnRH antagonists did not interfere with the process of ovulation, sperm migration to the site of fertilization, or early embryo development. In additional experiments with mice, GnRH antagonists inhibited in vitro fertilization. One fertilization event that was specifically inhibited by GnRH antagonists was the process of sperm binding to the zona pellucida. This step was precisely monitored using the hemizona assay. GnRH antagonists did not affect sperm movement or acrosomal status. These observations indicated that local treatment with GnRH antagonists inhibit in vivo fertilization and give additional support to the idea that endogenous GnRH may play an important role during fertilization by increasing the efficiency of sperm-zona binding.     

6-Bicyclopiperazinyl-1-arylsulfonylindoles and 6-bicyclopiperidinyl-1-arylsulfonylindoles derivatives as novel, potent, and selective 5-HT6 receptor antagonists

A novel series of 6-bicyclopiperazinyl-1-arylsulfonylindoles and 6-bicyclopiperidinyl-1-arylsulfonylindoles derivatives was synthesized and found to be potent and selective 5-HT6 receptor antagonists.     

Inhibitory Effect of Calmodulin Antagonists and Calcium Antagonists on Fertilization-Induced Cyanide-Insensitive Respiration in Sea Urchin Eggs

During initial several minutes after fertilization, sea urchin eggs exhibited high rate of respiration which was only slightly inhibited by cyanide. This cyanide-insensitive respiration was inhibited by calcium antagonists, diltiazem and verapamil, and calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalensulfonamide hydrochloride (W-7), N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) and chlorpromazine, which were added within 1 min after insemination. The inhibitory effect of W-7 on cyanide-insensitive respiration was higher than that of W-5. Cyanide-sensitive respiration of fertilized eggs observed after this initial period was not inhibited by these compounds. Ca²⁺ influx in eggs just after fertilization was inhibited by calcium antagonists but was rather enhanced by calmodulin antagonists. Fertilization-induced stimulation of cyanide-insensitive respiration probably results from calmodulin-dependent reactions which are activated by Ca²⁺ influx.     

Central administration of peptide and small molecule MC4 receptor antagonists induce hyperphagia in mice and attenuate cytokine-induced anorexia

We investigated the effect of melanocortin 4 receptor (MC4) antagonists on food intake in mice. Food intake during the light phase was significantly increased by ICV administration of mixed MC3/MC4 antagonists (AgRP and SHU9119) or MC4 selective antagonist peptide [(Cyclo (1-5)[Suc-D-Nal-Arg-Trp-Lys]NH2] (MBP10) and the small molecule antagonists THP and NBI-30. Both mixed and selective antagonists significantly reversed anorexia induced by ICV administration of the MC4 agonist (c (1-6) HfRWK-NH2) and the cytokine IL-1beta. These findings provide pharmacological evidence that the MC4 receptor mediates the effects of melanocortin agonists and antagonists on food intake in mice, and support the idea that selective small molecule MC4 antagonists may be useful as therapeutics for cachexia.     

1-Phenyl-8-azabicyclo[3.2.1]octane ethers: a novel series of neurokinin (NK1) antagonists

1-Phenyl-8-azabicyclo[3.2.1]octane ethers are NK(1) receptor antagonists. Substitution at the 6-exo-position led to high affinity NK(1) antagonists with a prolonged duration of action in vivo. Incorporation of an alpha-methyl substituent in the pendent benzyl ether side chain gave compounds with increased selectivity over the hERG channel.     

Guanine nucleotides modulate the affinity of antagonists at beta-adrenergic receptors

Investigation of the properties of the binding of the radiolabelled antagonists (125I)-iodohydroxybenzylpindolol, (125I)-iodopindolol, and (125I)-iodocyanopindolol to beta-adrenergic receptors of L6 myoblast membranes revealed that guanine nucleotides caused a 2 to 4.5 fold increase in the apparent affinity of these antagonists. No significant effects of GTP were observed on the density of binding sites determined with each radioligand. GTP, GDP, and GMPPNP were of similar high affinity in producing this effect, while GMP was much less potent, and ATP was without effect. Under similar assay conditions GTP reduced the apparent binding affinity of the agonist isoproterenol for the beta-adrenergic receptors of L6 cells. The results indicate that, contrary to previous observations, guanine nucleotides affect not only the interactions of agonists with beta-adrenergic receptors, but also the interaction of antagonists with these adenylate cyclase-linked receptors.     

Procognitive 5-HT6 antagonists in the rat forced swimming test: Potential therapeutic utility in mood disorders associated with Alzheimer's disease

Aims5-HT₆ receptor subtype is predominantly expressed in the brain, and preclinical evidence suggests its potential role in the cognitive function. Brain microdialysis studies demonstrated that 5-HT₆ antagonists enhance not only cholinergic but also monoaminergic neurotransmission, a property that may differentiate from acetylcholine esterase (AChE) inhibitors such as donepezil. In this study we compared the antidepressant-like effects of 5-HT₆ antagonists with donepezil to determine whether their different effects on monoamines are behaviorally relevant.Main methodsSelective 5-HT₆ antagonists (SB-399885 and SB-271046) and donepezil were evaluated in the rat forced swimming test since this is known to identify drugs such as antidepressants which can increase brain monoamine levels. Binding assay was undertaken by using [¹²⁵I]SB-258585 to measure brain 5-HT₆ receptor occupancy.Key findingsSystemic administration of SB-399885 (3 and 10 mg/kg, i.p.) and SB-271046 (10 and 30 mg/kg, i.p.) produced a significant reduction of immobility time in the rat forced swimming test with a similar profile in terms of 5-HT₆ receptor occupancy (62 and 96% for 3 and 10 mg/kg SB-399885 respectively; 56 and 84% for 10 and 30 mg/kg SB-271046 respectively). In contrast, donepezil (0.5 and 1 mg/kg i.p.) did not show any effects in this model.SignificanceThese data suggest that 5-HT₆ antagonists, at doses corresponding to those occupy central 5-HT₆ receptors, could have an antidepressive effect in humans. This may differentiate 5-HT₆ antagonists from AChE inhibitors with respect to the mood control in the symptomatic treatment of Alzheimer's disease.     

Synthesis and biological activity of NK-2 selective tachykinin antagonists containing D-tryptophan

By introducing D-Trp in position 6 and 8 along with pyroglutamic acid (Pyr) in position 4 or Nle in position 10 of NKA(4-10) we have obtained selective although weak NK-2 tachykinin receptor antagonists. Similar substitutions, previously reported on the sequence of SP, gave rise to nonselective antagonists presumably for the limited selectivity of the agonist used as template. Further modifications like the addition of a third D-Trp in position 9 gave rise to more potent but less selective antagonists, thus showing that each amino acid substitution can dramatically affect selectivity.     

6-(2-Furanyl)-9H-purin-2-amine derivatives as A2A adenosine antagonists

Structure-activity relationships have been investigated through substitutions at the 9-position of the 2-amino-6-(2-furanyl) purine (5) to identify novel and selective A(2A) adenosine receptor antagonists. Several potent and selective antagonists were identified. In particular, compounds 20, 25, and 26 show very high affinity with excellent selectivity.     

The action of agonists and antagonists at the histamine H1 receptor and receptor protection studies in guinea pig ileum

We have examined the ability of histamine and the competitive reversible antihistamines to protect the histamine H1 receptor against alkylation with the 2-haloalkylamines, phenoxybenzamine and SY-14. In isolated guinea pig ileum these irreversible antagonists produce a parallel shift in the dose-response curve to histamine with retention of the maximum response if they are used at concentrations less than about 10(-6)M. Treatment with these 2-haloalkylamines in the presence of a high concentration of histamine did not alter the blocking activity. Thus histamine appears to be unable to protect its own receptor against irreversible blockade. The competitive reversible antagonists, on the other hand, did provide effective protection against irreversible blockade. It is likely that the competitive reversible H1 receptor antagonists have at least some part of their attachment site in common with irreversible antagonists of the 2-haloalkylamine type, while the inability of histamine to provide self-protection suggests that its primary attachment site is different from that of the antagonists.