SAR of a series of 5,6-dihydro-(9H)-pyrazolo[3,4-c]-1,2,4-triazolo[4,3-alpha]pyridines as potent inhibitors of human eosinophil phosphodiesterase.

The potency and physical properties of a previously reported 7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine series of human eosinophil phosphodiesterase inhibitors were improved by tying the lactam moiety into a triazolo ring. The resulting 5,6-dihydro-(9H)-pyrazolo[3,4-c]-1,2,4-triazolo[4,3-alpha]pyridine series provided nonionizable analogs with melting point properties suitable for micronization. Substitution at the 3-position of the 5,6-dihydro-(9H)-pyrazolo[3,4-c]-1,2,4-triazolo[4,3-alpha]pyridine tricycle led to a 2-thienyl analog, 19 (tofimilast), a potent PDE4 inhibitor with low oral bioavailability and no emesis-associated behaviors in ferrets at plasma concentrations up to 152 ng/mL.

A new chemical tool for exploring the role of the PDE4D isozyme in leukocyte function.

Nicotinamide (2) is a potent and selective inhibitor of the PDE4D isozyme and as a chemical tool selectively blocks eosinophil mediator release and chemotaxis thus linking the role of PDE4D to eosinophil function.

Discovery of CP-199,330 and CP-199,331: two potent and orally efficacious cysteinyl LT1 receptor antagonists devoid of liver toxicity.

CP-199,330 (3) and CP-199,331 (4) are cysLT1 receptor antagonists that are equipotent to marketed cysLT1 receptor antagonists zafirlukast and pranlukast, show good pharmacokinetics in rats and monkeys, and are devoid of liver toxicity in monkeys as seen in CP-85,958 (1).

Development of 2,2-dimethylchromanol cysteinyl LT1 receptor antagonists.

A new series of cysLT1 receptor antagonists represented by CP-288,886 (7) and CP-265,298 (8) were developed which are equipotent to clinical cysLT1 receptor antagonists Zafirlukast (1) and Pranlukast (2).

N-carbamoyl analogs of Zafirlukast: potent receptor antagonists of leukotriene D4.

Exploration of the indole nitrogen region of Zafirlukast (1) has uncovered a potent series of cysteinyl leukotriene D4 (LTD4) antagonists. These studies showed that a variety of functionality could be incorporated in this region of the molecule without sacrificing potency. Efforts to exploit this site in order to improve oral efficacy are discussed.

Development of new chromanol antagonists of leukotriene D4.

By addressing the issues of potency and metabolism in 3, a new series of LTD4 antagonists represented by (+)-26 was developed which is equipotent to clinical LTD4 antagonists Zafirlukast (1) and Pranlukast (2).

Isolation and characterization of PDE9A, a novel human cGMP-specific phosphodiesterase.

We have cloned and characterized the first human isozyme in a new family of cyclic nucleotide phosphodiesterases, PDE9A. By sequence homology in the catalytic domain, PDE9A is almost equidistant from all eight known mammalian PDE families but is most similar to PDE8A (34% amino acid identity) and least like PDE5A (28% amino acid identity). We report the cloning of human cDNA encoding a full-length protein of 593 amino acids, including a 261-amino acid region located near the C terminus that is homologous to the approximately 270-amino acid catalytic domain of other PDEs.

Isolation and characterization of PDE8A, a novel human cAMP-specific phosphodiesterase.

As part of our efforts to understand the regulation of intracellular cAMP and to generate new targets for pharmacological intervention, we have cloned and characterized the first isozyme in a new family of cyclic nucleotide phosphodiesterases, PDE8A. PDE8A is most similar to PDE4 (38.5% amino acid identity in the catalytic domain), but is clearly not a member of any of the seven known PDE families.

Tenidap inhibits 5-lipoxygenase product formation in vitro, but this activity is not observed in three animal models.

Objective And Design: The effect of tenidap on the metabolism of arachidonic acid via the 5-lipoxygenase (5-LO) pathway was investigated in vitro and in vivo.

Materials And Treatment: In vitro (cells). Arachidonic acid (AA) stimulated rat basophilic leukemia, (RBL) cells; A23817 activated neutrophils (human rat, and rabbit), macrophages (rat), and blood (human).

Biarylcarboxylic acids and -amides: inhibition of phosphodiesterase type IV versus [3H]rolipram binding activity and their relationship to emetic behavior in the ferret.

In addition to having desirable inhibitory effects on inflammation, anaphylaxis, and smooth muscle contraction, PDE-IV inhibitors also produce undesirable side effects including nausea and vomiting. In general, compounds that inhibit PDE-IV also potently displace [3H]rolipram from a high-affinity binding site in rat cortex. While this binding site has not been identified, it has been proposed to be an allosteric binding site on the PDE-IV enzyme.

Azelastine inhibits acute allergic dyspnea in a conscious guinea pig asthma model.

A simple, noninvasive, bias-flow ventilated wholebody plethysmographic technique and noninvasive pulmonary analyzer (Buxco dyspnea monitor) were used to quantitate allergic dyspnea in chronically sensitized freely moving guinea pigs. In this study, the effect of azelastine on aeroallergen-induced dyspnea in allergic guinea pigs was investigated. Aeroallergen challenge produced severe dyspnea which was characterized by a 390% increase in the amplitude of pseudo flow signal, a 93% increase in box pressure (delta P) and a 68% decline in relaxation time; these changes signify a tremendous increase in the effort of breathing.

Vortex flow filtration of mammalian and insect cells.

The use of vortex flow filtration for harvesting cells or conditioned medium from large scale bioreactors has proven to be an efficient, low shear method of cell concentration and conditioned medium clarification. Several 8-10 L batches of the human histiocytic lymphoma U-937 cell line (ATCC CRL 1593) were concentrated to less than 1 L by vortex flow filtration through a 3.0 microns membrane.

Allergic bronchial eosinophilia: a therapeutic approach for the selection of potential bronchial anti-inflammatory drugs.

Aeroallergen-induced infiltration of eosinophils in the bronchoalveolar lavage fluid (BALF) in guinea pigs was used as a marker of bronchial inflammation. Drugs were administered orally 4 h after aeroallergen challenge. Allergic bronchial eosinophilia in guinea pigs was inhibited by orally administered dexamethasone and methylprednisolone.

Aeroallergen-induced dyspnea in freely moving guinea pigs: quantitative measurement by bias flow ventilated whole body plethysmography.

Guinea pigs were sensitized and boostered with i.p. injections of ovalbumin (OA) 10 micrograms + Al(OH)3 100 mg.

Antigen-mediated pulmonary eosinophilia in immunoglobulin G1-sensitized guinea pigs: eosinophil peroxidase as a simple specific marker for detecting eosinophils in bronchoalveolar lavage fluid.

Eosinophil peroxidase (EPO) has been used previously to detect the number of eosinophils in the peritoneal exudate and bone marrow of mice. The present study was undertaken to determine 1) whether EPO activity may provide a measure of a change in eosinophils in bronchoalveolar lavage fluid (BALF) of guinea pigs, 2) whether immunoglobulin (Ig)G1 could play a role in pulmonary eosinophilia and 3) effects of pharmacological agents on the EPO response in an IgG1 passively sensitized animal model. The activity of EPO was assessed by the ability of cell lysates (0.

Characterization of aeroallergen-induced dyspnea in unrestrained guinea pigs by bias-flow-ventilated whole body plethysmography.

Aeroallergen-induced dyspnea in guinea pigs was associated with an increase in amplitude in the box pressure fluctuations (212%) and pseudo-flow signal (604%) and an 80% decline (from 0.19 to 0.04 s) in relaxation time (the time it takes the box pressure signal to drop from its peak to 1/3 of its peak value).

Anti-IL-5 monoclonal antibody inhibits allergic late phase bronchial eosinophilia in guinea pigs: a therapeutic approach.

In this study the effect of purified rat anti-mouse IL-5 monoclonal antibody on aeroallergen-induced infiltration of eosinophils in the bronchoalveolar lavage fluid of guinea pigs was studied. The i.p.

Effect of azelastine on activation and release stages of allergic histamine secretion in rabbit basophils.

In this study the effect of azelastine on the activation (antigen-dependent, Ca(2+)-dependent, Stage I) and release (antigen-independent, Ca(2+)-dependent, Stage II) phases of allergen-induced histamine secretion in rabbit mixed leukocytes (basophils) was investigated. Azelastine (5 microM, 10-min) and diltiazem (5 microM: a Ca2+ antagonist, 10 min) inhibited ragweed extract-induced histamine secretion during the Stage II (release) phase. Theophylline (100 microM), a phosphodiesterase inhibitor, added immediately before antigen challenge, inhibited allergic histamine secretion during the Stage I (activation) phase.

Evidence that peptidoleukotriene is a prerequisite for antigen-dependent thromboxane synthesis in IgG1-passively sensitized guinea pig lungs.

Lungs from guinea pigs passively sensitized with an affinity-purified IgG1 antibody produce both leukotriene (LT)D4 and thromboxane (Tx)B2 upon ex vivo antigen challenge. This study was undertaken to determine the possibility of endogenously generated peptido-LTs being a prerequisite for Tx synthesis. In immunoglobulin G1-sensitized lungs, exogenous LTD4 induced TxB2 production with a median effective dose of 4.

Inhibition of 5-HETE, LTB4, and LTC4 formation by azelastine in rat mixed peritoneal cells.

Azelastine produced a concentration-dependent inhibition of calcium ionophore A23187 (0.2 microM) stimulated generation of 5-HETE, leukotriene B4, and leukotriene C4 in rat mixed peritoneal cells, yielding IC50 values of 35.5, 47.

Antigen-dependent leukotriene synthesis and histamine release from IgG1 passively-sensitized guinea pig lungs ex vivo: relationship between serum levels of antigen-specific IgG1 and mediator synthesis/release.

Naive guinea-pigs were passively sensitized with varying amounts of affinity column purified, homologous, anti-ovalbumin IgG1 (anti-OA IgG1) and then examined for a) the capacity of lung tissue to release mediators (histamine and LTB4/LTD4) in response to antigen-challenge ex vivo and b) the attendant circulating levels of anti-OA IgG1. Intraperitoneal administration of anti-OA IgG1 (0.125-0.

Inhibition of PAF-induced histamine secretion and bronchoconstriction by WEB 2086.

Platelet activating factor (PAF) stimulates histamine secretion from rabbit mixed leukocytes in a concentration-dependent fashion (EC50 10 ng/ml). The PAF (10 ng/ml)-induced histamine secretion was inhibited by WEB 2086 (IC50 = 20 nM). Furthermore, PAF (10 ng/kg, i.

Involvement of peptidoleukotrienes in antigen-dependent thromboxane (TX) synthesis in IgG1-sensitized guinea pig lungs.

Lungs from IgG1-sensitized guinea pigs synthesize both leukotrienes (LTs) and thromboxane (Tx) upon ex vivo antigen challenge. This study was undertaken to investigate whether antigen-dependent Tx synthesis could result from prior formation of LTD4. In IgG1-sensitized lungs, LTD4 effectively induced Tx formation (ED50 = 2-4 nM).

Inhibition of allergic and nonallergic leukotriene C4 formation and histamine secretion by azelastine: implication for its mechanism of action.

Azelastine, an orally effective antiasthmatic agent, has been reported to inhibit antihistamine-resistant, leukotriene-mediated allergic bronchoconstriction in guinea pigs. This suggests that azelastine might act through inhibition of leukotriene (LT) C4/D4 synthesis. We have examined the effect of azelastine on allergic and nonallergic histamine secretion and LTC4 formation.

Inhibition of allergic histamine release from rat peritoneal mast cells by azelastine. Interaction with selected antiasthmatic drugs.

The in vitro interaction of azelastine (a new antiallergic/antiasthmatic drug) with albuterol (a beta 2 bronchodilator), theophylline (a phosphodiesterase inhibitor), disodium cromoglycate (DSCG, a mast cell-stabilizing agent) and prednisolone (a steroid) was studied for effects on allergic histamine release from rat peritoneal mast cells (RPMCs). The RPMCs preincubated with albuterol, theophylline, DSGC (10 min) and prednisolone (2h) caused a 2- to 18-fold decrease in the IC30 for azelastine. Significant potentiation (synergism) was seen only with albuterol (0.