Pulmonary Pharmacology (1992) 5, 137-141 PULMONARY PHARMACOLOGY
Effect of Mequitamium Iodide (LG 30435) on Airway Microvascular Leakage in the Guinea-pig L . Abelli*, F . Nappi, A. Subissit, S. Manzini, A . Giachetti Department of Pharmacology, Menarini Ricerche Sud, Pomezia (Roma) and tDepartment of Pharmacology, Laboratori Guidotti, Pisa, Italy
SUMMARY: Intravenous administration of mequitamium iodide (LG 30435) prevented the increase of tracheobronchial vascular permeability induced either by antigen challenge or by exogenous histamine in the guinea-pig, while it was ineffective against PAF, serotonin or capsaicin . These findings indicate that mequitamium iodide selectively interferes with the effect of histamine on airway microvascular leakage, mediated by histamine H, receptors, and is more potent than diphenhydramine, mequitazine or astemizole . Histamine receptor antagonism is likely to be a major determinant of the antiallergic activity of the compound, although additional mechanisms may be involved .
INTRODUCTION
MATERIALS AND METHODS
Mequitamium iodide [LG 30435, 1-methyl-3-(1OHphenothiazine-10-ylmethyl)-1-azoniabicyclo(2 .2 .2) octane iodide] is a new bronchodilator agent with multiple sites of action . This quaternary derivative of the H,-histamine antagonist mequitazine is effective against bronchoconstriction induced by various agonists 1-3 and is endowed with antiallergic activity .' ,' The activity of mequitamium iodide against bronchoconstriction induced by a number of mediators implicated in asthma may be beneficial in the treatment of this multifactorial disease . The importance of plasma exudation in the airways in the pathogenesis of asthma has been recently emphasized' and the hypothesis has been advanced that clinical efficacy of antiasthma drugs may reflect antiinflammatory properties, among them an effect on vascular permeability .' Therefore, experiments have been performed to investigate the activity of mequitamium iodide in inhibiting microvascular leakage induced by antigen challenge or by various inflammatory mediators in the tracheo-bronchial region of urethane-anaesthetized guinea-pigs . A comparison was made with standard antihistamine drugs, such as diphenhydramine, mequitazine and astemizole, and with the antiallergic drug ketotifen fumarate .
Vascular permeability Male Dunkin-Hartley guinea-pigs (Rodentia, T.Pallavicina, BG, Italy) weighing 300-500 g were used in the experiments . Plasma protein extravasation was measured as Evans Blue leakage, according to previously described methods ." Briefly, the animals were anaesthetized with urethane (1 .6 g/kg s .c .) and the left jugular vein was cannulated for drug administration, allowing the animals to recover at least 30 min after surgery . In experiments with capsaicin, the animals, including controls, were artificially ventilated (50 strokes/min x 10-12 ml/kg) by means of a Basile 7025 Rodent Ventilator . In separate experiments, 3-4 weeks before the antigen challenge (ovalbumin 200 tg/kg i.v .), guinea-pigs were sensitized by i .p . administration of ovalbumin (1 µg) and aluminum hydroxide (100 mg) dissolved in 0 .5 ml of sterile saline .'"' Evans Blue was administered intravenously (20 mg/kg dissolved in 0 .9% NaCl saline containing 1000 I .U ./ml heparin) 5 min before intravenous histamine-2HC1 (500 nmol/kg), serotonin creatinine sulphate (1000 nmol/kg), ovalbumin (200 pg/kg), PAY (0 .18 nmol/kg dissolved in 0 .25% bovine serum albumin in saline) or capsaicin (500 nmol/kg dissolved in 5% ethanol in saline) . Five minutes later the animals were perfused via the thoracic aorta with saline (80 ml in 45 s) to wash the dye out of the vascular system .
* For correspondence at: Faculty of Sciences, University della Tuscia, via San Camillo de Lellis/Block D, 01100 Viterbo, Italy . 0952-0600/92/020137+05 $03.00/0
137
© 1992 Academic Press Limited
138
L . Abelli et al
Test compounds (diphenhydramine-HCI, mequitazine, astemizole, ketotifen fumarate, and mequitamium iodide) were administered intravenously 210 min before administration of inflammatory mediators . Times and dosages of test compounds (see Figs 1-2 and Table 3) were previously selected according to the onset of their antibronchospastic activity (A . Subissi, personal communication) . The caudal segment of the trachea and the main stem bronchi were excised, weighed and extracted in 4 ml of formamide (50 °C for 16 h) . Tissue content of Evans Blue was determined by fluorometry (620 nm EX, 680 nm EM ; Perkin-Elmer 512 DB) and expressed as ng/mg of wet tissue weight .
100
75
50
25
0 0
0 100
75
Drugs The following compounds were used : Evans Blue (C.I . Direct blue 53), ovalbumin (5 x cryst .), 5-hydroxytryptamine creatinine sulphate and diphenhydramineHCl from Serva (Heidelberg, Germany) ; formamide, urethane, heparin sodium salt, bovine serum albumin, histamine-2110 and capsaicin from Sigma (St Louis, MO, USA) ; PAF(C, g) from Bachem (Bubendorf, Switzerland) ; astemizole and aluminum hydroxide from Janssen (Beerse, Belgium) . Ketotifen fumarate was a kind gift from Dr Handley (Sandoz, East Hanover, NJ, USA) . Mequitamium iodide and mequitazine were synthesized at Guidotti Labs by Dr L . Turbanti .
50
25
0 0.01
0 .1
1 Dose (p.moV kg i .v.)
10
100
Fig . I Dose-response curves for the inhibitory effect of mequitamium iodide (0 .03-1 µmol/kg, 2 min before), mequitazine (0 .1-1 µmol/kg, 10 min before), astemizole (0 .1-3 µmot/kg, 10 min before) and diphenhydramine (3-30 µmot/kg, 7 min before) on histamine (500 nmol/kg i .v .) induced plasma protein extravasation in guinea-pig caudal trachea (A) and main bronchi (B) . Each point is the mean of five to eight observations . Standard error of the mean does not exceed 15% and has not been shown for clarity. (O) Mequitamium ; (∎) mequitazine; (0) astemizole ; ( •) diphenhydramine.
Statistical analysis All values are given as mean±SEM . Numerical results were analysed by means of the Student's t-test for unpaired data . Regression analysis was performed by means of the least squares method . ED, values and 95% confidence limits were calculated accordingly .
RESULTS Intravenous administration of histamine (500 nmol/ kg) induced vascular permeability in the guinea-pig tracheo-bronchial region (93 ± 9 and 94 ± 9 ng Evans Blue/mg of wet tissue weight, in trachea and bronchi, respectively; n = 10 observations) : this produced a seven to eight-fold increase of dye content over saline controls (11 ± 1 and 13 ± 1 ng/mg of wet tissue weight, in trachea and bronchi, respectively ; n = 10 animals ; P
Effect of Mequitamium Iodide (LG 30435) on Airway Microvascular Leakage in the Guinea-pig L . Abelli*, F . Nappi, A. Subissit, S. Manzini, A . Giachetti Department of Pharmacology, Menarini Ricerche Sud, Pomezia (Roma) and tDepartment of Pharmacology, Laboratori Guidotti, Pisa, Italy
SUMMARY: Intravenous administration of mequitamium iodide (LG 30435) prevented the increase of tracheobronchial vascular permeability induced either by antigen challenge or by exogenous histamine in the guinea-pig, while it was ineffective against PAF, serotonin or capsaicin . These findings indicate that mequitamium iodide selectively interferes with the effect of histamine on airway microvascular leakage, mediated by histamine H, receptors, and is more potent than diphenhydramine, mequitazine or astemizole . Histamine receptor antagonism is likely to be a major determinant of the antiallergic activity of the compound, although additional mechanisms may be involved .
INTRODUCTION
MATERIALS AND METHODS
Mequitamium iodide [LG 30435, 1-methyl-3-(1OHphenothiazine-10-ylmethyl)-1-azoniabicyclo(2 .2 .2) octane iodide] is a new bronchodilator agent with multiple sites of action . This quaternary derivative of the H,-histamine antagonist mequitazine is effective against bronchoconstriction induced by various agonists 1-3 and is endowed with antiallergic activity .' ,' The activity of mequitamium iodide against bronchoconstriction induced by a number of mediators implicated in asthma may be beneficial in the treatment of this multifactorial disease . The importance of plasma exudation in the airways in the pathogenesis of asthma has been recently emphasized' and the hypothesis has been advanced that clinical efficacy of antiasthma drugs may reflect antiinflammatory properties, among them an effect on vascular permeability .' Therefore, experiments have been performed to investigate the activity of mequitamium iodide in inhibiting microvascular leakage induced by antigen challenge or by various inflammatory mediators in the tracheo-bronchial region of urethane-anaesthetized guinea-pigs . A comparison was made with standard antihistamine drugs, such as diphenhydramine, mequitazine and astemizole, and with the antiallergic drug ketotifen fumarate .
Vascular permeability Male Dunkin-Hartley guinea-pigs (Rodentia, T.Pallavicina, BG, Italy) weighing 300-500 g were used in the experiments . Plasma protein extravasation was measured as Evans Blue leakage, according to previously described methods ." Briefly, the animals were anaesthetized with urethane (1 .6 g/kg s .c .) and the left jugular vein was cannulated for drug administration, allowing the animals to recover at least 30 min after surgery . In experiments with capsaicin, the animals, including controls, were artificially ventilated (50 strokes/min x 10-12 ml/kg) by means of a Basile 7025 Rodent Ventilator . In separate experiments, 3-4 weeks before the antigen challenge (ovalbumin 200 tg/kg i.v .), guinea-pigs were sensitized by i .p . administration of ovalbumin (1 µg) and aluminum hydroxide (100 mg) dissolved in 0 .5 ml of sterile saline .'"' Evans Blue was administered intravenously (20 mg/kg dissolved in 0 .9% NaCl saline containing 1000 I .U ./ml heparin) 5 min before intravenous histamine-2HC1 (500 nmol/kg), serotonin creatinine sulphate (1000 nmol/kg), ovalbumin (200 pg/kg), PAY (0 .18 nmol/kg dissolved in 0 .25% bovine serum albumin in saline) or capsaicin (500 nmol/kg dissolved in 5% ethanol in saline) . Five minutes later the animals were perfused via the thoracic aorta with saline (80 ml in 45 s) to wash the dye out of the vascular system .
* For correspondence at: Faculty of Sciences, University della Tuscia, via San Camillo de Lellis/Block D, 01100 Viterbo, Italy . 0952-0600/92/020137+05 $03.00/0
137
© 1992 Academic Press Limited
138
L . Abelli et al
Test compounds (diphenhydramine-HCI, mequitazine, astemizole, ketotifen fumarate, and mequitamium iodide) were administered intravenously 210 min before administration of inflammatory mediators . Times and dosages of test compounds (see Figs 1-2 and Table 3) were previously selected according to the onset of their antibronchospastic activity (A . Subissi, personal communication) . The caudal segment of the trachea and the main stem bronchi were excised, weighed and extracted in 4 ml of formamide (50 °C for 16 h) . Tissue content of Evans Blue was determined by fluorometry (620 nm EX, 680 nm EM ; Perkin-Elmer 512 DB) and expressed as ng/mg of wet tissue weight .
100
75
50
25
0 0
0 100
75
Drugs The following compounds were used : Evans Blue (C.I . Direct blue 53), ovalbumin (5 x cryst .), 5-hydroxytryptamine creatinine sulphate and diphenhydramineHCl from Serva (Heidelberg, Germany) ; formamide, urethane, heparin sodium salt, bovine serum albumin, histamine-2110 and capsaicin from Sigma (St Louis, MO, USA) ; PAF(C, g) from Bachem (Bubendorf, Switzerland) ; astemizole and aluminum hydroxide from Janssen (Beerse, Belgium) . Ketotifen fumarate was a kind gift from Dr Handley (Sandoz, East Hanover, NJ, USA) . Mequitamium iodide and mequitazine were synthesized at Guidotti Labs by Dr L . Turbanti .
50
25
0 0.01
0 .1
1 Dose (p.moV kg i .v.)
10
100
Fig . I Dose-response curves for the inhibitory effect of mequitamium iodide (0 .03-1 µmol/kg, 2 min before), mequitazine (0 .1-1 µmol/kg, 10 min before), astemizole (0 .1-3 µmot/kg, 10 min before) and diphenhydramine (3-30 µmot/kg, 7 min before) on histamine (500 nmol/kg i .v .) induced plasma protein extravasation in guinea-pig caudal trachea (A) and main bronchi (B) . Each point is the mean of five to eight observations . Standard error of the mean does not exceed 15% and has not been shown for clarity. (O) Mequitamium ; (∎) mequitazine; (0) astemizole ; ( •) diphenhydramine.
Statistical analysis All values are given as mean±SEM . Numerical results were analysed by means of the Student's t-test for unpaired data . Regression analysis was performed by means of the least squares method . ED, values and 95% confidence limits were calculated accordingly .
RESULTS Intravenous administration of histamine (500 nmol/ kg) induced vascular permeability in the guinea-pig tracheo-bronchial region (93 ± 9 and 94 ± 9 ng Evans Blue/mg of wet tissue weight, in trachea and bronchi, respectively; n = 10 observations) : this produced a seven to eight-fold increase of dye content over saline controls (11 ± 1 and 13 ± 1 ng/mg of wet tissue weight, in trachea and bronchi, respectively ; n = 10 animals ; P
