Treatment of hypertension with ketanserin,a new selective 5-HT2 receptor antagonist.

The new selective 5-HT2 receptor blocking agent ketanserin was given in a dose of 10 mg intravenously to 12 patients with essential hypertension. It caused a distinct fall in supine systemic arterial, right atrial, pulmonary artery, and pulmonary capillary "wedge" pressures. Cardiac output, renal blood flow, and glomerular filtration rate showed no persistent changes. Thus 5-HT2 receptor blockade caused dilatation of both resistance and capacitance vessels and of the renal vascular bed. Heart rate and plasma concentrations of renin and noradrenaline rose after ketanserin. These data suggest that 5-HT may have a role in maintaining high blood pressure.     

Stereoselective synthesis of a new muscarinic M3 receptor antagonist, J-104129.

A diastereoselective synthesis of J-104129 (1) was developed. A key step of this synthesis was Michael addition of enolate generated from cis-chiral dioxolane 2 to cyclopentenone, followed by hydrogenolysis of the resultant enol triflate 4. A mixture of cyclopentyldioxolane (5, 6) was hydrolyzed with sodium hydroxide to yield carboxylic acid 7 in 86% ee.     

Receptor-mediated autocrine growth-stimulatory effect of 5-hydroxytryptamine on cultured human pancreatic carcinoid cells.

5-hydroxytryptamine (5-HT) is a mitogen for fibroblasts, vascular smooth muscle cells, renal mesangial cells, and jejunal crypt cells. The human carcinoid cell line (termed BON) that we established in our laboratory from a pancreatic carcinoid tumor produces and secretes 5-HT. In this study, therefore, we examined the effect of 5-HT on growth of BON cells. Furthermore, by use of selective 5-HT receptor antagonists, we examined receptor and post-receptor mechanisms by which 5-HT-induced responses were produced. 5-HT stimulated growth of BON cells. 5-HT stimulated phosphatidylinositol (PI) hydrolysis in a dose-dependent fashion and inhibited cyclic AMP production in a dose-dependent fashion. The 5-HT1A/1B receptor antagonist, SDZ 21-009, prevented the reduction of cyclic AMP production evoked by 5-HT and inhibited the mitogenic action of 5-HT. The 5-HT1C/2 receptor antagonist, mesulergine, competitively inhibited PI hydrolysis, but did not affect the mitogenic action of 5-HT. The mitogenic action of 5-HT and the reduction of cyclic AMP production evoked by 5-HT were also inhibited by pertussis toxin. These results suggest that 5-HT is an autocrine growth factor for BON cells and that mitogenic mechanism of 5-HT involves receptor-mediated inhibition of the production of cyclic AMP which may be linked to pertussis toxin-sensitive GTP binding protein. 8-bromo-cyclic AMP inhibited growth of BON cells whereas 8-bromo-cyclic GMP had no effect on cell growth. Involvement of protein kinase A in BON cell growth regulation was confirmed by the observation that a cAMP-dependent protein kinase antagonist (Rp-cAMPS) could stimulate BON cell growth.     

Characterization of the human 5-hydroxytryptamine1B receptor.

We report the cloning of a human gene encoding the 5-hydroxytryptamine1B receptor. The receptor has the characteristics of a G-protein-linked receptor and is most homologous to the human 5-HT1D receptor. This human 5-HT receptor gene, most abundantly expressed in striatum, is localized on chromosome 6, at 6q13, and the gene encoding the 5-HT1D receptor is localized on chromosome 1. Radioligand studies indicate that the affinity of [3H]5-HT is 16 +/- 2 nM. Drug competition studies indicate that the receptor displays high affinity (i.e. less than 40 nM) for 5-HT, 5-carboxyamidotryptamine, methiothepin, and metergoline.     

Possible upregulation of hypothalamic 5-hydroxytryptamine receptors in patients with postviral fatigue syndrome.

OBJECTIVE--To study the dynamic function of hypothalamic 5-hydroxytryptamine receptors in patients with postviral fatigue syndrome. DESIGN--Prospective comparison of patients with postviral fatigue syndrome with two control groups. SETTING--Department of neurology, University of Glasgow, Southern General Hospital; department of psychiatry, St James''s Hospital, Dublin. SUBJECTS--15 patients with postviral fatigue syndrome, 13 age and sex matched healthy subjects, and 13 patients with primary depression. MAIN OUTCOME MEASURES--Serum prolactin concentrations before and one, two, and three hours after administration of buspirone. RESULTS--Because of the effects of sex hormones on prolactin secretion data for men and women were analysed separately. There was no significant difference in baseline prolactin concentrations between patients with postviral fatigue syndrome and healthy subjects or those with primary depression. However, the percentage difference between peak and baseline values was significantly higher in patients with postviral fatigue syndrome than the control groups (one way analysis of variance: women, p = 0.003; men, p = 0.004). CONCLUSIONS--The results suggest upregulation of hypothalamic 5-hydroxytryptamine receptors in patients with postviral fatigue syndrome but not in those with primary depression. The buspirone challenge test may therefore be useful in distinguishing these two conditions. Larger studies are required to explore the potential value of drugs acting on central 5-hydroxytryptamine receptors in the treatment of patients with the postviral fatigue syndrome.     

Implication of acidic lipids in 5-hydroxytryptamine receptor mechanisms

To establish the possible involvement of acidic lipids in 5-HT receptor mechanisms, we subjected whole rat brain synaptic plasma membranes to treatment with several kinds of lipid-modifying reagents and examined the [3H]5-HT and [3H]spiperone binding properties of the membranes. [3H]5-HT binding was decreased by treatment with Azure A, while [3H]spiperone binding was not altered. Similarly, prior treatment with arylsulphatase reduced the former binding, but had no effect on the latter binding. On the other hand, neither [3H]ligand binding was sensitive to phospholipases C and D. In contrast, prior treatment with phospholipase A2 (unheated) drastically decreased the [3H]5-HT binding and also affected the [3H]spiperone binding to some extent. Chelation of Ca2+ by EGTA (5 mM) prior to incubation of membranes with the unheated phospholipase A2 did not completely prevent the inhibitory effect of this enzyme on [3H]5-HT binding, while in the heated enzyme (at 100 degrees C for 10 min) EGTA exhibited this preventive effect perfectly. Furthermore, it was an interesting find that at least a low concentration of the heated phospholipase A2 (0.01 U) had no effect on the [3H]spiperone binding, as contrasted with the case of [3H]5-HT binding. In addition, the reduction of [3H]5-HT binding capacity in membranes treated with phospholipase A2 (heated and unheated) was restored only slightly by treatment with BSA (1%). Scatchard analysis of the [3H]5-HT binding showed that Azure A and phospholipase A2 (heated) decreased the Bmax values with no significant alteration in the KD values, whereas arylsulphatase increased only the KD value. All these observations infer that certain acidic lipids may play a role as the recognition site(s) or modulator(s) of 5-HT1 receptor molecules.     

SCH 57790: a novel M2 receptor selective antagonist

As a decrease in cholinergic neurons has been observed in Alzheimer's Disease (AD), therapeutic approaches to AD include inhibition of acetylcholinesterase to increase acetylcholine levels. Evidence suggests that acetylcholine release in the CNS is modulated by negative feedback via presynaptic M2 receptors, blockade of which should provide another means of increasing acetylcholine release. Structure-activity studies of [4-(phenylsulfonyl)phenyl]methylpiperazines led to the synthesis of 4-cyclohexyl-alpha-[4-[[4-methoxyphenyl]sulfinyl]-phenyl]-1-piperazin eacetonitrile. This compound, SCH 57790, binds to cloned human M2 receptors expressed in CHO cells with an affinity of 2.78 nM; the affinity at M1 receptors is 40-fold lower. SCH 57790 is an antagonist at M2 receptors expressed in CHO cells, as the compound blocks the inhibition of adenylyl cyclase activity mediated by the muscarinic agonist oxotremorine. This compound should be useful in assessing the potential of M2 receptor blockade for enhancement of cognition.     

Binding interactions of antagonists with 5-hydroxytryptamine3A receptor models

Homology modeling was performed on the N-terminal extracellular regions of human, mouse, and guinea pig 5-hydroxytryptamine type 3A receptors (5-HT3R) based on the 24% sequence homology with and on the crystal structure of the snail acetylcholine binding protein (AChBP). Docking of 5-HT3 antagonists granisetron, tropisetron, ondansetron, dolasetron ('setrons), and (+)-tubocurarine suggests an aromatic binding cleft behind a hydrophilic vestibule. Several intra- and interface interactions, H-bonds, and salt bridges stabilize the pentameric structure and the binding cleft. The planar rings of antagonists are intercalated between aromatic side-chains (W183-Y234, Y143-Y153). S227 donates H-bonds to the carbonyl groups of 'setrons. The tertiary ammonium ions interact with E236, N128 or E129, and/or W90 (cation-pi interaction). This offers a molecular explanation of the pharmacophore models of 5-HT3R antagonists. Docking artifacts suggest some ambiguities in the binding loops A and C of the 5-HT3AR models. Lower potencies of (+)-tubocurarine for human, and those of tropisetron for guinea pig 5-HT3ARs can be attributed to steric differences of I/S230 in the binding cleft and to distinct binding interactions with E229 and S227, respectively. Ligand binding interferes with crucial intra- and interface interactions along the binding cleft.     

Solution structure of a unique C5a semi-synthetic antagonist: implications in receptor binding.

The tertiary structure of a unique C5a receptor antagonist was determined by two-dimensional NMR spectroscopy. The core domain of this 8-kDa antagonist exists as an antiparallel helical bundle, similar to recombinant human (rh)-C5a. However, unlike C5a, the antagonist's C terminus was found to be conformationally restricted along a groove between helices one and four in the core domain. This conformational restriction situates C-terminal D-Arg 75 in a wedge between core residues Arg 46 and His 15. Correlation of the antagonist's tertiary structure with point mutation analysis revealed the formation of a positively charged contiguous contact surface comprised of D-Arg 75, Arg 46, Lys 49, and His 15. The significance of this surface in generating antagonist properties implies a single binding site with the C5a receptor and provides a structural template for drug design.     

Molecular cloning and characterization of a rat brain cDNA encoding a 5-hydroxytryptamine1B receptor.

To date, there have been at least eight different receptors for the neurotransmitter serotonin (5-HT) identified in the central nervous system. These receptors fall into four pharmacological classes: 5-HT1, 5-HT2, 5-HT3 and 5-HT4. The 5-HT1 class has been shown to contain at least four pharmacologically distinct subtypes, 5-HT1A-D. Of these, cDNAs encoding the 5-HT1A and 5-HT1C receptors have been previously characterized. We now report the cloning and expression of a rat brain cDNA encoding another member of the 5-HT1 receptor family. Transient expression of this clone demonstrated high-affinity binding of [3H]5-HT with a pharmacological profile corresponding to that of the 5-HT1B subtype: 5-CT, 5-HT greater than propranolol greater than methysergide greater than rauwolscine greater than 8-OH-DPAT. In situ hybridization revealed expression of cognate mRNA within cells of the dorsal and median raphe nuclei, consistent with previous reports that the 5-HT1B receptor acts as an autoreceptor on 5-HT terminals in this species. mRNA expression was also detected in cells within the CA1 region of hippocampus, striatum, layer 4 of cortex and in the cerebellum, suggesting a previously unrecognized post-synaptic role for the 5-HT1B receptor.     

J-104129, a novel muscarinic M3 receptor antagonist with high selectivity for M3 over M2 receptors

A new class of 4-acetamidopiperidine derivatives has been synthesized and investigated for human muscarinic receptor subtype selectivity. Introduction of a hydrocarbon chain of appropriate length into the piperidine nitrogen of the racemic N-(piperidin-4-yl)-2-cyclobutyl-2-hydroxy-2-phenylacetamide platform conferred up to 70-fold selectivity for human muscarinic M3 receptors over M2 receptors. Subsequent synthetic derivatizations resulted in highly potent M3 receptor antagonists with selectivity greater than two orders of magnitude for M3 over M2 receptors, from which the analogue 4r was selected. Preparation of both enantiomers of 4r led to the identification of (2R)-N-[1-(4-methyl-3-pentenyl)piperidin-4-yl]-2-cyclopentyl-2-hyd roxy-2-phenylacetamide (J-104129, (R)-4r), which exhibited 120-fold selectivity for M3 receptors (Ki = 4.2 nM) over M2 receptors (Ki = 490 nM). In isolated rat trachea, (R)-4r potently and specifically antagonized acetylcholine (ACh)-induced responses with a K(B) value of 3.3 nM. The highly subtype-selective profile was also seen in isolated rat tissue assays (50-fold) and in anesthetized rats (> 250-fold). Oral administration of J-104129 ((R)-4r) antagonized ACh-induced bronchoconstriction with an ED50 value of 0.58 mg/kg in rats. Thus, J-104129 ((R)-4r) may effectively facilitate bronchodilation in the treatment of obstructive airway disease.     

The 5-HT receptor antagonist M100,907 prevents extracellular glutamate rising in response to NMDA receptor blockade in the mPFC

We recently found that intracortical injection of the selective and competitive N-methyl-D-aspartate (NMDA) receptor antagonist 3-(R)-2-carboxypiperazin-4-propyl-1-phosphonic acid (CPP) impaired attentional performance in rats and blockade of 5-hydroxytryptamine (5-HT)2A receptors antagonized this effect. Here, we used the microdialysis technique in conscious rats to study the effect of CPP on extracellular glutamate (GLU) in the medial prefrontal cortex (mPFC) and the regulation of this effect by 5-HT2A receptors. Intraperitoneal injection of 20 mg/kg CPP increased extracellular GLU in the mPFC (201% of basal levels) but had no effect on 5-HT. Intracortical infusion of 100 microm CPP increased extracellular GLU (230% of basal values) and 5-HT (150% of basal values) in the mPFC, whereas 30 microm had no significant effect. The effect of 100 microm CPP on extracellular GLU was abolished by tetrodotoxin, suggesting that neuronal activity is required. Subcutaneous injection of 40 microg/kg M100,907 completely antagonized the effect of 100 microm cpp on extracellular GLU, whereas 10 microg/kg caused only partial attenuation. Likewise, intracortical infusion of 0.1 microm M100,907 completely reversed the increase of extracellular GLU induced by CPP. These findings show that blockade of NMDA receptors in the mPFC is sufficient to increase extracellular GLU locally. The increase of cortical extracellular GLU may contribute to CPP-induced cognitive deficits and blockade of 5-HT2A receptors may provide a molecular mechanism for reversing these deficits caused by dysfunctional glutamatergic transmission in the mPFC.     

Structural requirements of 5-hydroxytryptamine-moduline analogues to interact with the 5-hydroxytryptamine1B receptor

Using magnetic resonance methods and a clinically relevant rodent model of sepsis, we have made in vivo measurements of increased intracellular calcium in a pathologic state in the CNS. The intracellular calcium concentration was increased nearly twofold in septic rat brain compared with controls (p < 0.0001). This result, in a fully intact functioning mammalian system, ties together a previous spectrum of indirect evidence from numerous laboratories suggesting an important role for elevated intracellular calcium in sepsis. In addition, levels of the proinflammatory cytokine tumor necrosis factor-a were elevated threefold in septic rat brain (p < 0.02), and electron microscopic examination revealed scattered injury in approximately 0.25% of glial cells. These findings are discussed in light of the current understanding of the pathophysiology of sepsis.     

Cardioselective profile of AF-DX 116, a muscarine M2 receptor antagonist

AF-DX 116 (see chemical name below) is a competitive antagonist of muscarine receptors in peripheral organs. In contrast to pirenzepine, its behaviour in functional experiments indicates selectivity for the M2 muscarinic subtype. In pithed rats AF-DX 116 inhibits vagally-induced bradycardia, an M2 response, (ED50 32 micrograms/kg i.v.) in preference to the M1-mediated pressor response to McN-A-343 (ED50 211 micrograms/kg i.v.). AF-DX 116 further discriminates among M2 receptors, showing a high affinity for the cardiac muscarine receptors. In isolated preparations, AF-DX 116 has a tenfold higher affinity for the muscarine receptors of the heart (pA2 7.33) than for those in smooth muscles (pA2 6.39-6.44). The same profile appears from animal studies, where the compound is a more potent antagonist of either endogenously or exogenously activated cardiac muscarine responses as compared to vascular, smooth muscle or secretory responses. In general, the ratios of potencies (ED50) observed in cardiac vs. other muscarine mediated functions ranged between 30 and 50. Atropine showed no discrimination, inhibiting all muscarine responses in the same range of doses. In the conscious dog intravenous AF-DX 116 increased basal heart rate, and completely reversed the reflex bradycardia induced by clonidine. Tachycardia was dose-related (ED50 79 micrograms/kg i.v.), and occurred independently of background sympathetic tone. AF-DX 116 clearly distinguishes between M1- and M2-mediated responses; it also emphasizes the long-recognized heterogeneity among the peripheral M2 subtypes. AF-DX 116, for its pronounced cardioselectivity, may have a therapeutic potential in the treatment of sinus bradycardia.     

Preferential suppression of delayed-type hypersensitivity by L-156,602, a C5a receptor antagonist.

In the course of our screening for in vivo immunomodulating substances in which sheep red blood cells (SRBC) and heat-killed Brucella abortus cells (thymus dependent and independent antigens, respectively) for antibody production assays, and trinitrobenzene sulfonic acid (TNBS) for delayed-type hypersensitivity (DTH) assay were adopted as antigens, we detected a DTH-specific suppressive activity. The producing organism was isolated from a soil sample collected in Ushiku City, Ibaraki, Japan and identified with Streptomyces sp. A1502 (FERM P-12448). The active component was identified with L-156,602, a C5a receptor antagonist. L-156,602 suppressed both TNBS-induced and TNP-SRBC-induced DTH while it enhanced antibody production against SRBC, Brucella abortus, and TNP-SRBC. L-156,602 significantly suppressed DTH induced by direct injection of type 1 helper T cells and its relevant antigen into hind-footpads, indicating that the efferent phase of DTH was affected by L-156,602. The results demonstrated that L-156,602 preferentially suppressed the DTH response.     

DAU 6285: a novel antagonist at the putative 5-HT4 receptor.

The antagonistic properties of DAU 6285, an azabicycloalkyl benzimidazolone derivative, at putative 5-hydroxytryptamine4 (5-HT4) receptors were investigated in in vitro preparations of guinea-pig ileum and human atrium, in comparison to ICS 205-930. DAU 6285 behaved as a competitive antagonist in all the preparations examined. Its affinity (pA2) ranged between 6.50 and 7.12 in the test models considered. The affinity of ICS 205-930 was 2-3 fold lower. At variance with ICS 205-930, DAU 6285 displayed a weak affinity for 5-HT3 receptors (pKi = 6.1, rat cortex; pA2 less than 5, guinea-pig ileum). In the guinea-pig ileum, DAU 6285 (10 microM) did not exert antimuscarinic, antihistaminic, antinicotinic or myolytic activity. Moreover, it did not bind to other 5-HT receptor subtypes, or to adrenergic, dopaminergic, benzodiazepine, nicotine, GABA receptors. DAU 6285 may represent a suitable tool for studies in the field of 5-HT4 receptors.