Stephen Eckel

Biochemical characterization of desloratadine, a potent antagonist of the human histamine H1 receptor

We have characterized desloratadine (5H-benzo[5,6]cyclohepta[1,2-b]pyridine, 8-chloro-6,11-dihydro-11-(4-piperidinylidene), CAS 100643-71-8) as a potent antagonist of the human histamine H(1) receptor. [3H]Desloratadine bound to membranes expressing the recombinant human histamine H(1) receptor in Chinese hamster ovary cells (CHO-H(1)) in a specific and saturable manner with a K(d) of 1.1+/-0.2 nM, a B(max) of 7.9+/-2.0 pmol/mg protein, and an association rate constant of 0.011 nM(-1) x min(-1). The K(d) calculated from the kinetic measurements was 1.5 nM. Dissociation of [3H]desloratadine from the human histamine H(1) receptor was slow, with only 37% of the binding reversed at 6 h in the presence of 5 microM unlabeled desloratadine. Seventeen histamine H(1)-receptor antagonists were evaluated in competition-binding studies. Desloratadine had a K(i) of 0.9+/-0.1 nM in these competition studies. In CHO-H(1) cells, histamine stimulation resulted in a concentration-dependent increase in [Ca(2+)](i) with an EC(50) of 170+/-30 nM. After a 90-min preincubation with desloratadine, the histamine-stimulated increase in [Ca(2+)](i) was shifted to the right, with a depression of the maximal response at higher concentrations of antagonist. The apparent K(b) value was 0.2+/-0.14 nM with a slope of 1.6+/-0.1. The slow dissociation from the receptor and noncompetitive antagonism suggests that desloratadine may be a pseudoirreversible antagonist of the human histamine H(1) receptor. The mechanism of desloratadine antagonism of the human histamine H(1) receptor may help to explain the high potency and 24-h duration of action observed in clinical studies.

Phospholipase D in homogenates from HL-60 granulocytes: Implications of calcium and G protein control

Occupancy of chemotactic peptide receptors leads to rapid initiation of phospholipase D (PLD) activity in intact dimethylsulfoxide-differentiated HL-60 granulocytes (Pai, J.-K, Siegel, M.I., Egan, R.W., and Billah, M.M. (1988) J. Biol. Chem. 263, 12472). To gain further insight into the activation mechanisms, PLD has been studied in cell lysates from HL-60 granulocytes, using 1-0-alkyl-2-oleoyl-[32P]phosphatidylcholine (alkyl-[32P]PC), 1-0-[3H]alkyl-2-oleoyl-phosphatidylcholine [( 3H]alkyl-PC) and [14C]arachidonyl-phospholipids as substrates. In the presence of Ca2+ and GTP gamma S, post-nuclear homogenates degrade alkyl-[32P]PC to produce 1-0-alkyl-[32P]phosphatidic acid (alkyl-[32P]-PA), and in the presence of ethanol, also 1-0-alkyl-[32P]phosphatidylethanol (alkyl-[32P]PEt). By comparing the 3H/32P ratios of PA and PEt to that of PC, it is concluded that PA and PEt are formed exclusively by a PLD that catalyzes both hydrolysis and transphosphatidylation between PC and ethanol. Furthermore, PC containing either ester- or ether-linkage at the sn-1 position is degraded in preference to phosphatidylethanolamine and phosphatidylinositol by PLD in HL-60 cell homogenates. It is concluded that HL-60 granulocytes contain a PC-specific PLD that requires both Ca2+ and GTP for activation.

Pharmacological characterization of postjunctional α-adrenoceptors in human nasal mucosa

Background Functional α1- and α2-adrenoreceptor subtype pharmacology was characterized in an in vitro human nasal mucosa contractile bioassay. Methods Nasal mucosa was obtained from 49 donor patients and mucosal strips were placed in chambers filled with Krebs–Ringer solution and attached to isometric force transducers. Results Nonselective α-adrenoreceptor agonists epinephrine, norepinephrine, and oxymetazoline produced concentration-dependent contractions of isolated human nasal mucosa (pD2= 5.2, 4.9, and 6.5, respectively). The α2-adrenoreceptor agonist BHT-920 (10 μM)–induced contractions were blocked by yohimbine (0.01–1 μM) and prazosin (0.01–1 μM) inhibited the contractile response to the α1-adrenoreceptor agonist phenylephrine (10 μM). Histological analysis showed that phenylephrine and BHT-920 differentially contracted the arteries and veins of human nasal mucosa, respectively. Conclusion Our results indicate that functional α1- and α2-adrenoceptors are present and functional in human nasal mucosa. The a 2-adrenoceptors display a predominant role in contracting the veins and the α1-adrenoceptors appear to preferentially constrict the human nasal arteries.

Synergistic antiallergic activity of combined histamine H1- and cysteinyl leukotriene1-receptor blockade in human bronchus.

Mast cell histamine (HA) and cysteinyl leukotrienes (CysLT) account for most of the early phase bronchospasm in asthma. However, activation of the smooth muscle CysLT1-receptor plays a major role in asthmatic bronchospasms. CysLT-receptor antagonists or CysLT-synthesis inhibitors are efficacious in asthma but do not completely abolish asthmatic bronchospasms. A recent clinical study showed that combined antagonists loratadine (H1) and zafirlukast (CysLT1) were more effective against allergic bronchospasms than either drug alone. We examined the combined efficacy of H1- and CysLT1-receptor antagonists in allergic human bronchus. The H1- and CysLT1-receptor antagonists chlorpheniramine (CTM; 1 microM) and MK-571 (0.03 microM), were tested alone and in combination, against anti-human IgE antibody (Ab)-induced contractions of passively sensitized isolated human bronchus. Ab-induced allergic contractions were reduced 15% and 36% by CTM (1 microM) and MK-571 (0.03 microM), respectively. Combined CTM (1 microM) and MK-571 (0.03 microM) significantly inhibited the Ab response by 87%. Mechanistic investigations in isolated human bronchus and cultured human cord blood mast cells suggest that H1- and CysLT-receptor interactions likely occur at the airway smooth muscle level. CTM and MK-571 synergistically inhibited human allergic bronchospasm in the present in vitro model. The mechanism underlying this synergistic activity requires further investigation.

Structure-activity relationships of 3-substituted N-benzhydryl-nortropane analogs as nociceptin receptor ligands for the treatment of cough

A series of 3-axial-aminomethyl-N-benzhydryl-nortropane analogs have been synthesized and identified to bind to the nociceptin receptor with high affinity. Many of these analogs showed high binding selectivity over classic opioid receptors such as mu receptor. The synthesis and structure-activity relationships around the C-3 nortropane substitution are described. Selected compounds with potent oral antitussive activity in the guinea pig model are disclosed.

Relationship of phospholipase C- and phospholipase D-mediated phospholipid remodeling pathways to respiratory burst activation in human neutrophils stimulated by Candida albicans hyphae.

Neutrophil (PMN) oxidant release, a key component of defenses against disseminated candidiasis, was preceded by oxidant generation after stimulation by Candida albicans hyphae. Opsonized or unopsonized hyphae triggered phospholipase D (PLD) activation within 5 or 30 s, respectively, forming 1‐O‐alkyl‐phosphatidic acid (alkyl‐PA) or 1‐O‐alkyl‐phosphatidyl‐ethanol in the presence of ethanol. Ethanol, which competitively lowers phosphatidic acid (PA) production, caused dose‐dependent inhibition of superoxide (O2 ‐) generation after hyphal stimulation but altered neither baseline‐unstimulated O2 ‐ production nor responses to phorbol myristate acetate. PA rises evoked by unopsonized hyphae began 2 min before significant O2 ‐ release, also preceding both phospholipase C activation and cytosolic Ca2+ rises. Diacylglycerol (DAG) rose in two distinct phases after stimulation by opsonized or unopsonized hyphae, peaking briefly after 60 or 120 s, respectively, followed by prolonged secondary rises. Initial DAG rises preceded inositol triphosphate elevations evoked by unopsonized hyphae. Though PA rose before DAG, no dephosphorylation of PA to form 1‐O‐alkyl‐DAG was noted. Propranalol, which increases PA accumulation by inhibiting PA phosphohydrolase, lowered PMN O2 ‐ responses to hyphae. Early DAG rises temporally overlapped respiratory burst initiation but PMN responses to hyphae were unchanged by a DAG kinase inhibitor, R59022, which blocks phosphorylation of DAG to PA and enhances DAG accumulation. Thus, neither PA nor DAG accumulation individually accounted for triggering PMN O2 ‐ responses to hyphae. PLD activation and PA production may facilitate PMN fungicidal responses to hyphae but play an indirect role in initiating the respiratory burst. J. Leukoc. Biol. 57: 842–850; 1995.

Differential plasma duration of antiplatelet-activating factor and antihistamine activities of oral Sch 37370 in humans

Preclinical studies have established that Sch 37370 (1‐acetyl‐4‐(8‐chloro‐5,6‐dihyro‐11H‐benzo[5,6]‐cyclohepta[1,2‐b]pyridin‐11‐ylidene)piperidine) is an orally active antagonist of platelet‐activating factor (PAF) and histamine H1‐receptors with potential therapeutic use in the treatment of asthma. To evaluate the efficacy and duration of anti‐PAF and antihistamine actions of oral Sch 37370 in humans, a single dose (5 mg/kg) of Sch 37370 was given orally to each of 10 male subjects in a placebo‐controlled, double‐blind crossover study. Blood samples were drawn before and at various times (2 to 48 hours) after Sch 37370 or placebo. Plasma samples were analyzed for Sch 37370 by a gas chromatographic method, for the anti‐PAF activity by measuring the aggregation of platelets stimulated with PAF, and for the antihistamine activity by measuring displacement of [3H]pyrilamine from rat brain membrane binding sites. The plasma anti‐PAF activity declined from high levels at 2 hours to barely detectable levels at 24 hours; however, significant activity was still present at 12 hours. The plasma levels of Sch 37370 closely paralleled the anti‐PAF profile. The plasma antihistamine activity reached a maximum within 2 to 8 hours and declined thereafter. However, 48 hours after Sch 37370, the antihistamine activity was still present at a significant level in most subjects. It is concluded that, in humans, oral Sch 37370 antagonizes both PAF and histamine with plasma antihistamine activity lasting longer than plasma anti‐PAF activity.

Bronchoconstrictor effect of the tachykinin NK3-receptor agonists [MePhe7]-neurokinin B and senktide in the isolated guinea pig lung

ABSTRACT To determine whether bronchoconstriction can be mediated via the tachykinin NK3 receptors, isolated guinea pig lungs were challenged with the exogenous tachykinin NK3-receptor agonists [MePhe7]-neurokinin B ([MePhe7]-NKB) and senktide. [MePhe7]-NKB induced bronchoconstriction (EC50 = 11.8 ± 1.7 μM) that was significantly inhibited by the tachykinin NK3-receptor antagonist SB 223412 at 10 μM (EC50 = 24.4 ± 4.5 μM). Senktide also induced bronchoconstriction (EC50 = 96.2 ± 20.3 μM) and the bronchoconstriction was significantly reduced by SB 223412 at 1 and 10 μM (EC50 = 270.8 ± 78.9 μM and 388.3 ± 105.5 μM, respectively). Although the authors demonstrated that SB 223412, [MePhe7]-NKB, and senktide are potent and selective for the tachykinin NK3 receptors in binding and functional (Ca2+ mobilization) assays, the tachykinin NK1-receptor antagonist CP 99,994 at 1 μM (EC50 = 32.7 ± 8.5 μM) produced inhibition of [MePhe7]-NKB–induced bronchoconstriction, whereas the tachykinin NK2-receptor antagonist SR 48968 at 0.1 μM (EC50 = 213.2 ± 42.9 μM) blocked senktide-induced bronchoconstriction. These data suggest that [MePhe7]-NKB and senktide caused bronchoconstriction in guinea pig through activation of the tachykinin NK3-receptors but the tachykinin NK1- and/or NK2-receptors are also involved in the response.

Real-time assay of tryptase release from human umbilical cord blood-derived mast cells.

Mast cells are the central mediating cells of allergic reactions responding via the high-affinity IgE receptor (FcεRI) through binding of IgE and specific antigen (1). This activation leads to the release of preformed mediators stored in secretory granules (such as histamine, α and β tryptases, and chymase) as well as to the de novo synthesis of leukotrienes and prostaglandins and the production and release of various cytokines and chemokines (1). The monitoring of mediator release has been used in the past as a direct measurement of mast cell activation. In particular, assays to detect histamine have long been the standard used for studies of mast cell function (2–6). In addition, although the release of the mast cell protease tryptase has been used to monitor mast cell activation, those assays describe a multi-step technique in which tryptase levels are determined at a given endpoint after removal of cells (7,8). We report the development of a real-time assay for mast cell tryptase release from small numbers of cells (104 cells/assay point). This assay measures the release of tryptase from cells real-time and does not require manipulation of the cells other than incubation with appropriate stimuli. To propagate human cord blood-derived mast cells (CMBCs), human umbilical cord blood mononuclear progenitors (Poeitic Technologies, Gaithersburg, MD, USA) were placed into culture medium (RPMI/10% FBS; Invitrogen, Carlsbad, CA, USA) in the presence of stem cell factor and IL-6 (R&D Systems, Minneapolis, MN, USA), both at 100 ng/mL, at a density of 1 × 106 cells/mL (modification of Reference 9). One half of the culture medium was changed each week. After five weeks, IL-4 (R&D Systems) was introduced into the medium at 100 ng/mL. Cultures were continued for eight weeks and evaluated by staining with Toluidine blue dye. Cells used for functional assays were a minimum of 90% Toluidine blue positive. Eightto ten-week-old CBMCs were lysed by three rounds of freeze/ thaw, and the lysate was diluted in tryptase assay buffer (100 mM TrisHCl, pH 8.0, 100 mM NaCl) in a final volume of 50 μL and placed in the wells of a 96-well plate (Pierce Chemical, Rockford, IL, USA). An additional 50 μL of buffer alone or buffer containing protease inhibitors (all from Sigma St. Louis, MO, USA) was added to each well. A final 50 μL substrate [tosyl-glypro-lys-p-nitroanilide (10) (Sigma), 25 mM stock in 100% DMSO] in buffer was added to a final concentration of 500 μM, and the plate was immediately read in a plate reader (Molecular Devices, Sunnyvale, CA, USA) at 410 nm in the kinetic mode for 15 min with data collection every 30 s. Enzyme activity was determined as the rate of change of A due to cleavage of the substrate (velocity) and reported as mOD/min. The proteolytic activity present in varying numbers of cells against a serine protease substrate (tosyl-gly-pro-lys-p-nitro anilide) is shown in Figure 1 along with the activity of 5 nM rh-β-tryptase (Promega, Madison, WI, USA). Proteolytic activity above background could be detected from as few as 100 cells and was linear with cell number through the range tested (r2 = 0.99). This protease was inhibited by serine protease inhibitors (antipain, BABIM, benzamidine, leupeptin, and TLCK) and lactoferrin (which inhibits tryptase by competing for heparin) (data not shown) (11). Cysteine protease inhibitors (aprotinin, pepstatin, and TPCK) were not effective (data not shown). This pattern of inhibition suggests that the proteolytic activity being measured is indeed that of tryptase. To assay the real-time release of tryptase, CBMCs were sensitized for 24 h or longer in the presence of varying concentrations of human IgE (Biodesign, Saco, ME, USA). Before use, the cells were pelleted for 4 min at 1000× g and resuspended in RPMI at 2 × 105 cells/mL. Fifty microliters of cells (approximately 10 000 cells/well) were added to each well of a 96-well plate. Either 50 μL of buffer or buffer containing compounds to be tested was added to each well, and the plate was Benchmarks