[The respiratory system and safety pharmacology]. / Système respiratoire et pharmacologie de sécurité.

Many drugs affect respiration in man. The changes in respiratory function following their administration are due to a direct effect on the respiratory system or are the consequence of a central, metabolic (alteration of the acid-base balance) or vascular effect (pulmonary hypertension). Regulatory documents (CPMP, FDA and MHW drafts) are in agreement in considering the respiratory system as a vital function to explore during safety pharmacology studies. On the basis of these recommendations, the first studies to be performed should be on conscious unrestrained animals, in general the drug being administered as a single dose. The effects on the respiratory function are best studied by plethysmography in the guinea-pig or rat. The measurement of ventilatory parameters--respiratory rate, tidal volume, inspiratory time, expiratory time, peak inspiratory flow, peak exploratory flow and resistance--allow the differentiation between drugs affecting respiratory control and those altering lung mechanical properties. The pulmonary hypertension risk could be evaluated in the dog during haemodynamic studies. Finally, the study of the effects on blood gases should always be carried out in conscious animals, usually in the dog. For drugs belonging to pharmacological classes presenting a high respiratory risk, complementary studies should be considered. The extrapolation from healthy persons to ill patients (especially chronic respiratory insufficiency patients) of the incurred risk is often difficult. For this reason, it is very useful to study the effect of such drugs on the respiratory function of pathological animals.

Effect of limonene and sobrerol on monocrotaline-induced lung alterations and pulmonary hypertension.

The effects of two monocyclic monoterpenes, limonene and sobrerol, known as inhibitors of farnesyltransferase activity, were studied on monocrotaline (MCT)-induced lung injury, pulmonary hypertension and right ventricular hypertrophy in male Wistar rats. After 14 days, pulmonary arterial pressure values and the right ventricle to left ventricle plus septum weight ratios, RV/(LV + S), were markedly increased in rats subcutaneously injected with MCT (60 mg/kg). Limonene and sobrerol, administered daily at the oral dose of 400 mg/rat, markedly decreased the MCT-induced alterations. After treatment for 21 days, limonene still prevented pulmonary hypertension and the increase in RV/(LV + S). Both monoterpenes also reduced the increase in pulmonary arterial media thickness, the development of interstitial fibrosis and the increase in the number of macrophages in intra-alveolar spaces and of lymphocytes around the pulmonary veins. The present data indicate that treatment of rats with inhibitors of farnesyltransferase, like limonene and sobrerol, regulate the development of pulmonary hypertension.

Involvement of a phosphoramidon-sensitive endopeptidase in the processing of big endothelin-1 in the guinea-pig.

In anaesthetized and ventilated guinea-pigs, i.v. injection of 1 nmol/kg big endothelin-1 (big ET-1) did not evoke significant changes in pulmonary inflation pressure (PIP) and mean arterial blood pressure (MBP), whereas injection of the same dose of endothelin-1 (ET-1) induced marked and rapid bronchoconstrictor and pressor responses. Administered at the dose of 10 nmol/kg, big ET-1 provoked significant increases in PIP and MBP, which developed slowly and were long-lasting as compared to those evoked by ET-1. When big ET-1 was incubated for 45 min at 37 degrees C with alpha-chymotrypsin (2 mU/nmol) or pepsin (1 microgram/nmol) and then injected into guinea-pigs at the dose of 1 nmol/kg, marked bronchoconstrictor and pressor responses were observed, with kinetics similar to those noted after administration of the same dose of ET-1. The magnitude of the alpha-chymotrypsin- or pepsin-treated big ET-1 responses was similar to that induced by ET-1, incubated or not with the enzymes. Injected i.v. at the dose of 5 mg/kg, 5 min before the challenge, phosphoramidon almost totally inhibited the bronchoconstrictor and pressor responses induced by 10 nmol/kg big ET-1, whereas thiorphan (5 mg/kg) partially reduced the increase in PIP and exerted a minimal effect on the changes in MBP. Administered at the dose of 20 mg/kg per os, 1 h before i.v. administration of 10 nmol/kg big ET-1, enalapril maleate and captopril did not significantly alter the bronchoconstriction and the hypertensive response evoked by the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacologic modulation of picryl chloride-induced contact dermatitis in the mouse.

A biphasic response of ear swelling was observed 2 h and 24 h after application of the antigen to picryl chloride-sensitized Balb/c mice. A platelet-activating factor (PAF) antagonist, BN 52063, or the anti-inflammatory drug, betamethasone, applied topically or injected subcutaneously, inhibited in a dose-dependent fashion the antigen-induced increase in ear thickness observed after 24 h. In addition, BN 52063 and betamethasone presented a synergistic effect when administered in vivo simultaneously and subcutaneously. Indomethacin administered subcutaneously at the time of the antigen challenge significantly potentiated the early swelling phase and inhibited the late one. In contrast, the inhibitors of histamine and serotonin, ketotifen and methysergide, respectively, modulated mostly the early, and to a lower extent the late phase when administered at the time of antigen challenge. In contrast, none of these drugs inhibited the late phase reaction when administered 4 h after the antigen. A significant eosinophil and mononuclear-cell ear infiltrate was observed following topical application of the antigen, a phenomenon that was markedly reduced by either BN 52063 or betamethasone. These results demonstrate the effectiveness of PAF antagonists, either alone or in association with glucocorticosteroids, in experimental CD, the modulation of the infiltration of eosinophils and mononuclear cells possibly explaining part of the inhibitory action of these drugs.

Comparison of the effects of intra-arterial and aerosol administration of endothelin-1 (ET-1) in the guinea-pig isolated lung.

1. Intra-arterial injection of endothelin-1 (ET-1, 400 pmol; 1 microgram) in guinea-pig isolated perfused lungs, induced increases in pulmonary inflation pressure (PIP) and perfusion pressure (PPP), associated with oedema formation and thromboxane B2 (TxB2) release but not with the generation of sulphidopeptide leukotrienes or release of histamine. In contrast, aerosol administration of ET-1 (3, 6, 10 micrograms ml-1, for 2 min) evoked a dose-dependent increase in PIP, without significant changes in PPP, oedema formation or TxB2 release. 2. Addition of indomethacin (5 microM) or BW 755C (10 or 100 microM), but not nordihydroguaiaretic acid (NDGA, 50 microM) or FPL 55712 (10 microM), to the perfusion medium led to a significant inhibition of the increases in PIP and PPP, TxB2 release and oedema formation evoked by intra-arterial injection of 400 pmol ET-1. In contrast, indomethacin (5 microM), BW 755C (100 microM) or FPL 55712 (10 microM), added to the perfusion medium 10 min prior to challenge, did not affect the increase in PIP induced by a 2-min aerosol of a solution of ET-1 10 micrograms ml-1. 3. In vivo aerosol administration of indomethacin (100 mg ml-1, for 20 min) to non-anaesthetized guinea-pigs, 15 min before lung removal, did not modify the bronchopulmonary response evoked in isolated perfused lungs by an aerosol of ET-1 10 micrograms ml-1. However, under the same experimental conditions, indomethacin significantly inhibited TxB2 release evoked by aerosolized arachidonic acid (2 mg ml-1). 4. In conclusion, the present study shows that when injected by the intra-arterial route, ET-1 effects are mediated primarily via the generation of cyclo-oxygenase metabolites of arachidonic acid, whereas when the aerosol route is used, the peptide appears to act on airway smooth muscle cells, through an indomethacin-insensitive process which may involve some other, as yet unidentified, mediator(s).

Comparison of the bronchopulmonary and pressor activities of endothelin isoforms ET-1, ET-2, and ET-3 and characterization of their binding sites in guinea pig lung.

In anesthetized and ventilated guinea pigs intravenous injection of ET-1, ET-2, or ET-3 (1 or 2 nmol/kg) induced similar dose-dependent increases in pulmonary inflation pressure (PIP) associated with increases in mean arterial blood pressure (MBP). Pretreatment of the guinea pigs with 1 mg/kg intravenous indomethacin significantly inhibited the increase in PIP evoked by 2 nmol/kg of ET-1, ET-2, or ET-3. In contrast, the increase in MBP following injection of the various ET isotypes was not significantly affected by indomethacin. Injection of ET-1, ET-2, or ET-3 (40, 120, and 400 pmol) via the pulmonary artery of isolated and perfused guinea pig lungs induced dose-dependent increases in PIP and pulmonary perfusion pressure (PPP), thromboxane B2 (TXB2) release, and formation of lung edema. In keeping with the in vivo results, no marked differences were observed between the activities of ET-1, ET-2, and ET-3 on isolated and perfused guinea pig lungs. Indomethacin (5 microM) added to the perfusion medium significantly inhibited the alterations of PIP and PPP, TXB2 release, and edema formation evoked by 400 pmol ET-1, ET-2, or ET-3. High-affinity binding sites for ET-1, ET-2, and ET-3 exhibiting similar characteristics were identified on guinea pig lung membrane. Therefore ET-1, ET-2, and ET-3 exert comparable bronchopulmonary and pressor activities in the guinea pig and probably act via interaction with the same binding site. In addition, the ET-induced increase in PIP and pulmonary vasoconstriction are primarily mediated via the production of cyclooxygenase metabolites.

Effect of long-term infusion of platelet-activating factor on pulmonary responsiveness and morphology in the guinea-pig.

The effect of chronic administration of platelet-activating factor (PAF) on airway reactivity, cell recruitment and lung morphology in the guinea-pig has been investigated. Alzet osmotic minipumps delivering either PAF (7.2 mg/kg/14 days) in 0.25% (w/v) bovine serum albumin in saline (saline-BSA), acetylcholine or saline-BSA alone were implanted s.c. in the neck region of guinea-pigs and connected to the jugular vein. In some experiments, implanted and non-implanted animals were treated daily with the PAF antagonist, BN 52021 (15 mg/kg, twice a day, p.o.). On day 15 after minipump implantation, the animals were anesthetized with urethane (1.2 g/kg, i.p.) and tracheal cannula was inserted for mechanical ventilation. Pulmonary inflation pressure (PIP) was monitored and airway responsiveness was assessed by administration of increasing doses of histamine (0.2-100 micrograms/kg, i.v.). As compared to saline-BSA-treated or non-implanted guinea-pigs, chronic treatment of the animals with PAF induced a significant (p less than 0.01) increase in airway response. No significant change in airway responsiveness was observed following chronic acetylcholine administration. In contrast, regardless of the treatment of the animals, no change in the threshold dose of histamine inducing alteration in PIP was noted, suggesting that PAF induces bronchopulmonary hyperreactivity rather than hyperresponsiveness. In addition, no significant difference was observed in the in vitro responsiveness to histamine of lung parenchymal strips from animals having received PAF or saline-BSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Bronchopulmonary and pressor activities of endothelin-1 (ET-1), ET-2, ET-3, and big ET-1 in the guinea pig.

In anesthetized and ventilated guinea pigs, intravenous injection of ET-1, ET-2, or ET-3 induced similar rapid and dose-related increases in pulmonary inflation pressure (PIP) and mean arterial blood pressure (MABP). Indomethacin inhibited the increase in PIP evoked by ET-1, ET-2, or ET-3, whereas the changes in MABP following injection of the various ET isotypes were not significantly affected. Injection of ET-1, ET-2, or ET-3 via the pulmonary artery of isolated guinea pig lungs induced similar dose-dependent increases in PIP and pulmonary perfusion pressure (PPP), thromboxane B2 (TxB2) release, and formation of lung edema. Indomethacin (5 microM), added to the perfusion medium, significantly inhibited the alterations of PIP, PPP, TxB2 release, and lung edema formation evoked by the three ET isoforms. Intravenous injection of 1 nmol/kg of big ET-1 to guinea pigs did not induce significant changes in PIP and MABP. When administered at a dose of 10 nmol/kg, big ET-1 provoked marked slow-developing and sustained increases in PIP and MABP. When big ET-1 was incubated in vitro with either alpha-chymotrypsin or pepsin and injected into guinea pigs at a dose of 1 nmol/kg, marked rapid bronchoconstrictor and pressor responses were observed. The present results demonstrate that ET-1, ET-2, and ET-3 exert comparable bronchopulmonary and pressor activities in the guinea pig. On the contrary, big ET-1 exhibits moderate direct bronchoconstrictor and pressor effects and its hydrolysis by proteases appears to be essential for expression of its full activity.

Phosphoramidon potentiates the endothelin-1-induced bronchopulmonary response in guinea-pigs.

We investigated the effect of the endopeptidase-inhibitor, phosphoramidon, on the bronchopulmonary response induced by endothelin-1 in vivo or in isolated perfused lungs. In vivo aerosol administration of 1 or 3 ?g/ml endothelin-1 for 2 min provoked no significant bronchopulmonary response. When awake animals were pretreated by an aerosol of phosphoramidon (0.1 mM, for 15 min), the bronchopulmonary response induced by 1 and 3 ?g/ml endothelin-1 was markedly enhanced. In isolated guinea-pig lungs, aerosol administration of endothelin-1 (3 ?g/ml, for 2 min) evoked a low increase in pulmonary inflation pressure. Treatment of awake animals with an aerosol of phosphoramidon before lung recollection led to a significant potentiation of the endothelin-1-induced increase in pulmonary inflation pressure. These results demonstrate that phosphoramidon potentiates the in vivo and in vitro bronchopulmonary response evoked by low doses of endothelin-1 and suggest that endopeptidase-like enzymes present in the airway tissue modulate the effect of the peptide.

Bronchopulmonary and pressor effects of big-endothelin-1 in the guinea-pig.

Injection of 1 nmol/kg Big-endothelin-1 (ET-1) into anaesthetized and ventilated guinea-pigs did not evoke significant changes in pulmonary inflation pressure and mean arterial blood pressure. In contrast, injection of 1 nmol/kg ET-1 induced marked and rapid bronchoconstrictor and pressor responses. When administered at a dose of 10 nmol/kg, Big-ET-1 induced marked long-lasting changes in pulmonary inflation pressure and mean arterial blood pressure developing slowly as compared to those evoked by ET-1. Furthermore, these increases reached maximal values by 20 min for pulmonary inflation pressure and 45 min for mean arterial blood pressure after injection of the peptide. When Big-ET-1 was incubated with ?-chymotrypsin [45 min at 37 degrees C, enzyme : substrate ratio (wt/wt) : 0.5%] and injected into guinea-pigs at a dose of 1 nmol/kg, marked bronchoconstrictor and pressor responses were observed, developing with the same kinetics as those evoked by ET-1. The extent of the pressor response was similar and the bronchoconstriction was slightly lower than those evoked upon injection of 1 nmol/kg ET-1 treated or not with ?-chymotrypsin. The present results indicate that Big-ET exhibits moderate, if any, direct bronchoconstrictor and pressor activities in the guinea-pig. The slow metabolism of Big-ET-1 in an active form probably explains its long-lasting effects at a dose of 10 nmol/kg. This is indirectly confirmed by the in vitro treatment of Big-ET-1 with ?-chymotrypsin which converts the peptide into an active form.

Bronchopulmonary and vascular effect of endothelin in the guinea-pig.

Intravenous (i.v.) injection of endothelin in the anesthetized and ventilated guinea-pig induced a dose-dependent increase in pulmonary inflation pressure. The 1 nmol/kg dose rapidly enhanced pulmonary inflation pressure, reaching a peak at 30 s, as well as produced a marked and sustained increase in mean arterial blood pressure. A maximal increase in arterial blood pressure was observed 4 min after the injection of 1 nmol/kg endothelin and remained at a plateau for 20 min. The increase in pulmonary inflation pressure induced by endothelin was significantly enhanced by treatment of the guinea-pigs with propranolol (1 mg/kg i.v.). In contrast to what was observed in untreated guinea pigs, the injection of 1 nmol/kg endothelin in propranolol-treated animals produced within 2 min an irreversible and dramatic decrease in mean arterial blood pressure with a further decline for up to 90 min. The increase in pulmonary inflation pressure induced by 0.5 nmol/kg endothelin was also potentiated significantly in propranolol-treated guinea-pigs as compared to untreated animals. Endothelin 0.5 nmol/kg caused only a transient and non-significant increase in mean arterial blood pressure in both propranolol-treated and untreated animals. Injection of endothelin (40, 120 and 400 pmol) via the pulmonary artery into isolated guinea-pig lungs evoked significant increases in pulmonary inflation pressure and perfusion pressure accompanied by the dose-dependent release of TXB2 but, in contrast, no release of histamine. The in vitro effect of endothelin on pulmonary inflation pressure and perfusion pressure was potentiated when propranolol (1 microM) was added to the perfusion medium. These results demonstrate that the bronchopulmonary effects of endothelin are, at least in part, dissociated from the vascular action of the peptide.

Modification of allergen-induced airway obstruction and airway hyperresponsiveness in an allergic rabbit model by the selective platelet-activating factor antagonist, BN 52021.

Allergen challenge of eight ragweed-immunized rabbits elicited a prolonged airway obstruction that was evident during the 6-hour study period. Airway responsiveness to histamine was enhanced 24 hours after allergen exposure (geometric mean of the 50% reduction in effective dose [ED50] of dynamic compliance [Cdyn] before challenge, 3.46 mg/ml, decreased to 0.52 mg/ml after challenge, p less than 0.01; pulmonary resistance ED50 geometric mean before challenge, 3.50 mg/ml, decreased to 0.65 mg/ml after challenge, p less than 0.05). In a random crossover study, the selective platelet-activating factor antagonist, BN 52021 (60 mg/kg, orally, 1 hour before allergen challenge), significantly reduced the allergen-induced airway obstruction (p less than 0.005). In addition, pretreatment with BN 52021 reduced the associated increased airway responsiveness induced by allergen exposure (prechallenge Cdyn ED50, 1.93; after challenge, 2.13 mg/ml; pulmonary resistance ED50, 1.91 mg/ml before challenge and 1.69 mg/ml after challenge). These present observations suggest a role for platelet-activating factor in the pathogenesis of allergen-induced airway obstruction and in the development of airway hyperresponsiveness in an allergic rabbit model.

Action of platelet-activating factor (PAF) antagonists on the bronchopulmonary effects of PAF in the guinea-pig.

Intravenous injection of BN 52021 in anesthetized guinea-pigs, 5 min before challenge, inhibited in a dose-dependent fashion with an IC50 of 0.90 mg/kg the bronchoconstriction induced by PAF (60 ng/kg i.v.). However, BN 52021 did not prevent the leukopenia following PAF injection but significantly inhibited the thrombocytopenia induced by PAF. The dioxolan compound, BN 52111, dose-dependently reduced the bronchoconstriction (IC50 = 0.27 mg/kg) and at doses higher than 1 mg/kg partially antagonized the decrease in the number of circulating platelets and leukocytes induced by PAF. BN 52115 also markedly inhibited the bronchoconstriction (IC50 = 0.36 mg/kg) as well as the thrombocytopenia induced by PAF, but was without significant effect on the leukopenia. These results demonstrate that the two dioxolan compounds, BN 52111 and BN 52115, are more potent than BN 52021 in inhibiting the in vivo bronchopulmonary alterations induced by PAF. Since these alterations are related to the activation of platelets by the autacoid, these blood elements are probably the targets of BN 52111 and BN 52115. Injection of PAF (10 and 100 ng) via the pulmonary artery of ventilated and perfused guinea-pig lungs induced dose-dependent increases in pulmonary inflation pressure (PIP) and pulmonary perfusion pressure (PPP), associated with a dose-dependent release of thromboxane B2 (TxB2). Addition of BN 52021, BN 52111 or BN 52115 (0.1, 1 or 10 microM) to the perfusion medium, 15 min before challenge, dose-dependently inhibited the bronchopulmonary effects of PAF. Although BN 52111 was the more potent in inhibiting the PAF-induced increase in PIP, BN 52021 was the more active with respect to the PAF-evoked generation of TxB2, suggesting that the two phenomena are not directly related.

Effect of cyclosporin A and the platelet-activating factor (PAF) antagonist, BN 52021, on PAF- and antigen- induced bronchoconstriction in the guinea-pig.

The effects of the PAF antagonist, BN 52021, and cyclosporin A (CsA), either alone or in combination, on PAF- and antigen- induced bronchoconstriction were investigated in control and passively sensitized guinea-pigs, respectively. Although single administration of CsA alone has no effect on the PAF-induced bronchoconstriction, a marked inhibition of this phenomenon is observed when the drug is given along with an inactive dose of BN 52021. This effect of the association of the two drugs on the bronchoconstriction is also related to an action on the PAF-induced alterations in the number of leukocytes and platelets. In addition, administration of CsA for 48 hrs, which alone does not influence PAF-induced bronchoconstriction, markedly increases the inhibition evoked by BN 52021. Although bolus administration of CsA has no effect on the antigen -induced bronchoconstriction, a marked inhibition of this phenomenon is observed when the drug is given for 2 days. This inhibition by CsA is not further enhanced when the animals are also treated with BN 52021. These results strengthen the hypothesis that PAF and the immune system are involved in the regulation of bronchopulmonary reactions.

Effect of endothelin-1 on blood pressure and bronchopulmonary system of the guinea pig.

The bronchopulmonary and pressor effects of endothelin-1 (ET-1), a newly described vasoconstrictor peptide produced by endothelial cells, were investigated in the guinea pig. Intravenous injection of ET-1 (1 nmol/kg) induced an increase in pulmonary inflation pressure (PIP) as well as an important and sustained increase in arterial blood pressure (BP). Pretreatment of these animals with propranolol (1 mg/kg i.v.), provoked a significant enhancement of the ET-1-induced increase in PIP, accompanied by a dramatic and significant decrease of BP. When administered by aerosol for 1 min, ET-1 (1, 5, or 10 micrograms/ml) induced a dose-dependent increase in PIP that was maximal by 4-5 min, but no significant change of BP. Pretreatment of guinea pigs with propranolol (1 mg/kg), mepyramine (1 mg/kg i.v.), nifedipine (50 mg/kg i.p.), or verapamil (0.3 mg/kg i.v.) did not inhibit the bronchopulmonary response evoked by aerosol administration of 10 micrograms/ml of ET-1. In contrast, pretreatment of the animals with indomethacin (10 mg/kg i.v.) or BN 52021 (10 mg/kg i.v.) significantly reduced the bronchopulmonary response of ET-1 given by aerosol. Injection of ET-1 (0.1, 0.3, and 1 micrograms) into isolated guinea pig lungs caused significant increases in PIP that were accompanied by the release of TxB2 but not histamine. These results demonstrate that ET-1 induces bronchopulmonary alterations in the guinea pig that appear to be dissociated from the systemic vascular effects of the peptide.

Effect of long-term administration of platelet-activating factor on pulmonary responsiveness and morphology in the guinea pig.

To further substantiate the possible role of platelet-activating factor (PAF) in the development of bronchial hyperreactivity, Alzet osmotic minipumps loaded with the autacoid or solvent alone were placed under the back skin of male Hartley guinea pigs and connected to the jugular vein for 15 days. As compared to the animals treated with solvent alone, guinea pigs receiving PAF (20 micrograms/kg/h, 15 days) exhibited a hyperresponsiveness to histamine that was not observed in those simultaneously treated with PAF and the PAF antagonist BN 52021. Lungs from PAF-treated animals were congestive in appearance, bronchi and bronchioli were contracted, Reisseisen muscles were markedly hypertrophied, and a muciparous metaplasia of the endothelium was observed. The number of eosinophils was significantly increased in the parenchyma, as was the number of mast cells in the peribronchial zones in the lungs from guinea pigs chronically treated with PAF. Lungs from control animals did not show morphologic alterations or eosinophil and mast cell infiltration. These data indicate that long-term treatment with PAF induces profound alterations in the respiratory system, resembling those observed in asthmatic patients. This result is in keeping with the possible role of this autacoid in the development of bronchial hyperreactivity.

[Effect of long-term administration of platelet activating factor on pulmonary response and morphology in the guinea pig]. / Effet de l'administration chronique du "platelet activing factor" sur la réponse et la morphologie pulmonaires chez le cobaye.

The possible role of PAF in the development of bronchial hyperreactivity was investigated using Alzet osmotic minipumps loaded with PAF or solvent alone and implanted in male Hartley guinea-pigs. Compared to animals treated with solvent alone, those receiving PAF (20 micrograms/kg/h for 15 days) exhibited a hyperreactivity to histamine. The lungs from PAF-treated were congestive in appearance, bronchi and the bronchioli were contracted, Reisseissen muscles were markedly hypertrophied and a muciparous metaplasia of the epithelium was observed. In addition, the number of eosinophils was significantly increased in the parenchyma, as was the number of mast cells in the peribronchial regions. These data indicate that long term-treatment with PAF induces profound alterations in the respiratory system resembling those observed in asthmatic patients. They also strengthen the also strengthen the role of PAF in the development of bronchial hyperreactivity.