John Pillar

Inhibition of calcium ionophore (A23187)-stimulated histamine release from rat peritoneal mast cells by azelastine: Implications for its mode of action

Azelastine is a novel, orally effective, long-acting, antiallergic agent. The ability of azelastine to influence calcium ionophore A23187-induced histamine release from rat peritoneal mast cells was investigated and compared with selected antiallergic drugs. The concentrations of drugs required to inhibit A23187 (0.2 microM)-stimulated histamine release by 50% (IC50S, microM) were as follows: azelastine 5; diphenhydramine 52; and ketotifen 200. Theophylline and sodium cromoglycate in a concentration range of 0.1-1000 microM failed to exert any significant inhibition of histamine release. The inhibitory effects of azelastine on A23187-stimulated histamine release were antagonized by high concentrations of exogenous Ca2+ ions. These data suggest that azelastine inhibits A23187-stimulated histamine release by interfering with the influx of Ca2+ into the mast cells.

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. injection of anti-IL-5 antibody 4 h after aeroallergen challenge inhibited eosinophil infiltration in a dose-dependent fashion. The resulting ED50 was 10 (3.4-32.8) micrograms/kg. The clinical therapeutic usefulness of anti-IL-5 or anti-IL-5-producing cells in asthma/allergy treatment remains to be an intriguing possibility.

Inhibition by azelastine of nonallergic histamine release from rat peritoneal mast cells.

The ability of azelastine and selected antiallergic drugs to inhibit compound 48/80-induced and PS-potentiated, Con A-induced histamine release from RPMC was investigated. Azelastine, ketotifen, theophylline, and DSCG added simultaneously with the secretagogues or preincubated with the RPMC for 10 min before the addition of secretagogues produced concentration-dependent inhibition of histamine release. In general, the relative order of potency at calculated IC50 level was as follows: azelastine greater than ketotifen greater than theophylline greater than DSCG. The preincubation of RPMC with azelastine for 10 min exerted 3.5 times greater inhibition of Con A plus PS-stimulated histamine release but did not influence the inhibitory activity on compound 48/80-induced release. The duration of preincubation did not influence the inhibitory effects of ketotifen with either secretagogue. Theophylline and DSCG exerted significantly greater inhibition when they were added simultaneously with Con A plus PS. The inhibitory activity of DSCG was also significantly improved upon simultaneous addition with compound 48/80. These data demonstrated that azelastine is the most potent inhibitor of nonallergic histamine release from RPMC among the four antiallergic drugs examined.

Inhibition of allergic histamine release by azelastine and selected antiallergic drugs from rabbit leukocytes

The ability of azelastine to inhibit allergic histamine release from rabbit mixed leukocytes was studied and compared with selected antiallergic drugs. Azelastine, ketotifen, diphenhydramine, theophylline and disodium cromoglycate (DSCG) produced concentration-dependent inhibition of allergic histamine release from rabbit basophils. The concentrations inhibiting histamine release by 50% (IC50; microM) were as follows: azelastine = 4.5; ketotifen = 9.5; diphenhydramine = 18.9; theophylline = 56.9; DSCG = greater than 1,000. DSCG was added to the cells immediately prior to antigen challenge. All other drugs were preincubated for a period of 10 min prior to antigen challenge. At the IC50 level, azelastine is about 2, 4, 13 and greater than 200 times as effective as ketotifen, diphenhydramine, theophylline and DSCG, respectively. The IC50 of azelastine following 0, 10 and 30 min preincubation were 2.4, 1.9 and 3.5 microM, respectively. These observations showed: (1) azelastine is capable of acting rapidly on basophils and of inhibiting allergic histamine secretion, and (2) the prolongation of the preincubation time of azelastine up to 30 min with rabbit leukocytes did not exhibit any sign of tachyphylaxis (loss of activity). In conclusion, azelastine is a potent inhibitor of allergic histamine secretion from the leukocytes of ragweed-sensitized rabbits.

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. Azelastine and the known 5-lipoxygenase inhibitors, nordihydroguaiaretic acid and AA-861, exerted concentration-dependent inhibition of allergic LTC4 formation in chopped lung tissue from actively sensitized guinea pigs and calcium ionophore A23187-stimulated LTC4 synthesis in mixed peritoneal cells from rats. Azelastine also produced concentration-dependent inhibition of allergic and nonallergic histamine secretion from rat peritoneal mast cells. The ability of azelastine to inhibit allergic and nonallergic histamine secretion and LTC4 generation may contribute to its mode of action and its therapeutic efficacy.

In vitro inhibition of allergic histamine release by calcium antagonists

The ability of calcium entry blockers to inhibit allergic histamine release from rabbit leukocytes was studied. Bepridil, verapamil, nifedipine, diltiazem and TMB-8 produced concentration-dependent inhibition of allergic histamine release from rabbit leukocytes. The calculated IC50s (microM) were as follows: verapamil = 1.3; bepridil = 2.3; TMB-8 = 3.0; nifedipine = 3.3; and diltiazem = 5.3. Verapamil also exerted concentration-dependent inhibition of allergic histamine release from human basophils with an IC50 of 3.7 microM. These agents may act by interfering with the influx of Ca2+ into the leukocytes as well as calcium-dependent steps (e.g., activation of calmodulin, phospholipase A2 and/or 5-lipoxygenase etc.) in the process of histamine secretion.

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 (0.01, 0.1 and 1.0 microM, 10 min) and theophylline (1.0 microM, 10 min). The pre-exposure of RPMCs with DSCG (0.1, 1.0 and 10 microM) for a period of 10 min produced a slight leftward shift of azelastines concentration-effect curve (0.01, 0.1 and 10 microM added immediately before antigen challenge). This effect was not dependent on the concentration of DSCG. These data demonstrated (1) the lack of cross-tachyphylaxis between DSCG and azelastine, and (2) the synergistic interaction between azelastine and albuterol or theophylline.

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, 47.4, and 31.7 microM, respectively. Nordihydroguaiaretic acid (a potent 5-lipoxygenase inhibitor) also exerted a strong and concentration-dependent inhibition of 5-HETE and leukotriene B4 and C4 formation with IC50 values of 0.15, 0.09, and 0.1 microM, respectively. The inhibition of the formation of the products of the lipoxygenase pathway of arachidonic acid metabolism by azelastine may contribute to its overall antiallergic, antiasthmatic, and pulmonary anti-inflammatory activities.