Identification of a selective nonpeptide antagonist of the anaphylatoxin C3a receptor that demonstrates antiinflammatory activity in animal models.

The anaphylatoxin C3a is a potent chemotactic peptide and inflammatory mediator released during complement activation which binds to and activates a G-protein-coupled receptor. Molecular cloning of the C3aR has facilitated studies to identify nonpeptide antagonists of the C3aR. A chemical lead that selectively inhibited the C3aR in a high throughput screen was identified and chemically optimized. The resulting antagonist, N(2)-[(2,2-diphenylethoxy)acetyl]-L-arginine (SB 290157), functioned as a competitive antagonist of (125)I-C3a radioligand binding to rat basophilic leukemia (RBL)-2H3 cells expressing the human C3aR (RBL-C3aR), with an IC(50) of 200 nM. SB 290157 was a functional antagonist, blocking C3a-induced C3aR internalization in a concentration-dependent manner and C3a-induced Ca(2+) mobilization in RBL-C3aR cells and human neutrophils with IC(50)s of 27.7 and 28 nM, respectively. SB 290157 was selective for the C3aR in that it did not antagonize the C5aR or six other chemotactic G protein-coupled receptors. Functional antagonism was not solely limited to the human C3aR; SB 290157 also inhibited C3a-induced Ca(2+) mobilization of RBL-2H3 cells expressing the mouse and guinea pig C3aRS: It potently inhibited C3a-mediated ATP release from guinea pig platelets and inhibited C3a-induced potentiation of the contractile response to field stimulation of perfused rat caudal artery. Furthermore, in animal models, SB 290157, inhibited neutrophil recruitment in a guinea pig LPS-induced airway neutrophilia model and decreased paw edema in a rat adjuvant-induced arthritis model. This selective antagonist may be useful to define the physiological and pathophysiological roles of the C3aR.

SB 239063, a p38 MAPK inhibitor, reduces neutrophilia, inflammatory cytokines, MMP-9, and fibrosis in lung.

The effects of a second generation p38 mitogen-activated protein kinase (MAPK) inhibitor, SB 239063 [trans-1-(4-hydroxycyclohexyl)-4-(4-fluorophenyl)-5-(2-methoxypyridim idi n-4-yl)imidazole; IC(50) = 44 nM vs. p38 alpha], were assessed in models that represent different pathological aspects of chronic obstructive pulmonary disease (COPD) [airway neutrophilia, enhanced cytokine formation and increased matrix metalloproteinase (MMP)-9 activity] and in a model of lung fibrosis. Airway neutrophil infiltration and interleukin (IL)-6 levels, assessed by bronchoalveolar lavage 48 h after lipopolysaccharide (LPS) inhalation, were inhibited dose dependently by 3-30 mg/kg of SB 239063 given orally twice a day. In addition, SB 239063 (30 mg/kg orally) attenuated IL-6 bronchoalveolar lavage fluid concentrations (>90% inhibition) and MMP-9 activity (64% inhibition) assessed 6 h after LPS exposure. In guinea pig cultured alveolar macrophages, SB 239063 inhibited LPS-induced IL-6 production (IC(50) of 362 nM). In a bleomycin-induced pulmonary fibrosis model in rats, treatment with SB 239063 (2.4 or 4.8 mg/day via osmotic pump) significantly inhibited bleomycin-induced right ventricular hypertrophy (indicative of secondary pulmonary hypertension) and increases in lung hydroxyproline synthesis (indicative of collagen synthesis and fibrosis). Therefore, SB 239063 demonstrates activity against a range of sequelae commonly associated with COPD and fibrosis, supporting the therapeutic potential of p38 MAPK inhibitors such as SB 239063 in chronic airway disease.

Differences in time-related cardiopulmonary responses to hypoxia in three rat strains.

The cardiopulmonary profile of three rat strains (Sprague-Dawley, Wistar and High altitude-sensitive) was compared upon exposure to hypoxia (9% O2) for 0, 7 or 14 days. No differences were observed among the in vitro contractile (ET-1) and relaxant (carbachol) responses of pulmonary artery isolated from the three strains during normoxia. Chronic hypoxia decreased ET-1 contractile responses and diminished relaxant responses to carbachol similarly in all strains. In Sprague-Dawley, Wistar and High altitude-sensitive rats, pulmonary arterial pressure rose time-dependently and was elevated by 108%, 116% and 167%, respectively, after 14 days of hypoxia compared to normoxic controls. Right ventricular hypertrophy was increased by 51%, 93% and 55%, respectively, at 14 days. Hypoxia-induced hypertrophy and medial thickening in the pulmonary vasculature were more pronounced in High altitude-sensitive rats. Sprague-Dawley exhibited hypoxia-induced airway hyperresponsiveness to intravenous methacholine, but there were no hypoxia- or strain-related differences in in vitro tracheal contractility. Although each strain exhibited greater sensitivity for a particular hypoxia-induced parameter, pulmonary vascular functional and structural changes suggest that High altitude-sensitive rats represent a choice model of hypoxia-induced pulmonary hypertension.

SB 239063, a potent p38 MAP kinase inhibitor, reduces inflammatory cytokine production, airways eosinophil infiltration, and persistence.

The anti-inflammatory/antiallergic activity of a novel second-generation p38 mitogen-activated protein kinase inhibitor, SB 239063[trans-1-(4-hydroxycyclohexyl) -4-(4-fluorophenyl)-5-(2-methoxypyridimidin-4-yl)imidazole], was investigated in vivo and in vitro. SB 239063 had an IC(50) of 44 nM for inhibition of recombinant purified human p38alpha. In lipopolysaccharide-stimulated human peripheral blood monocytes, SB 239063 inhibited interleukin-1 and tumor necrosis factor-alpha production (IC(50) values = 0.12 and 0.35 microM, respectively). A role for p38 kinase in cytokine-associated inflammation in the mouse was shown by p38 activation in the lung and inhibition of lipopolysaccharide-induced tumor necrosis factor-alpha production by SB 239063 (ED(50) = 5.8 mg/kg p.o.). Antiallergic activity was demonstrated by essential abolition (approximately 93% inhibition) of inhaled ovalbumin (OA)-induced airway eosinophilia by SB 239063 (12 mg/kg p.o.), measured by bronchoalveolar lavage (BAL) in OA-sensitized mice. In addition, p38 kinase was found by Western analysis to be activated in guinea pig lung. Administration of SB 239063 (10 or 30 mg/kg p.o.) in conscious guinea pigs markedly reduced ( approximately 50% inhibition) OA-induced pulmonary eosinophil influx, measured by BAL 24 h after antigen. SB 239063 (10 mg/kg b.i.d. p.o.) administered after leukotriene D(4) inhalation, reduced by 60% the persistent airway eosinophilia seen at 4 days. Apoptosis of cultured eosinophils isolated from guinea pig BAL was increased by SB 239063 (1-10 microM) in the presence of interleukin-5. These results indicate that SB 239063 is a potent inhibitor of inflammatory cytokine production, inhibits eosinophil recruitment, in addition to enhancing apoptosis of these cells. Collectively, the results support the potential utility of p38 kinase inhibitors, such as SB 239063, for the treatment of asthma and other inflammatory disorders.

Effect of SB 217242 on hypoxia-induced cardiopulmonary changes in the high altitude-sensitive rat.

The effects of SB 217242, a non-peptide endothelin (ET) receptor antagonist, were investigated against hypoxia-induced cardiopulmonary changes in high altitude-sensitive rats. In isolated pulmonary artery rings, SB 217242 (30 n m) antagonized ET-1-induced contractions with a p KB of 8.0. There was no difference in the sensitivity to ET-1 or the potency of SB 217242 in pulmonary artery from normoxic rats vs. rats exposed to hypoxia (9% O2) for 14 days. However, there was a marked reduction in the maximum response to ET-1, but not to KCl or phenylephrine, in pulmonary artery from hypoxic rats; this phenomenon was inhibited by treatment of animals with SB 217242 (10.8 mg/day, ip by osmotic pump) for the 14-day hypoxic period. Furthermore, there was a significant reduction in carbachol-induced, endothelium-dependent relaxation of precontracted pulmonary artery from hypoxic animals; SB 217242 treatment during the hypoxic period did not influence this difference. Vehicle-treated rats exposed to 14-day hypoxia had 173% higher pulmonary artery pressures and 75% higher right/left+septum ventricular mass ratios compared to normoxic animals. SB 217242 (3.6 or 10.8 mg/day, ip) markedly reduced (80 and 95%, respectively) hypoxia-induced increases in pulmonary artery pressure. Right ventricular hypertrophy was inhibited by 40% at the 10.8 mg/day dose. Marked medial thickening and luminal stenosis of small and medium-sized pulmonary arteries was observed in hypoxic rats. The SB 217242-treated, hypoxia-exposed rats had comparable small and medium-sized arteries to normoxic rats. Rats treated with SB 217242 (10.8 mg/day) for the last 14 days of a 28-day hypoxic exposure had significantly lower pulmonary artery pressures than those of vehicle-treated rats. In addition, the effects of the selective ETA receptor antagonist, SB 247083, and the selective ETB receptor antagonist, A-192621 (3.6 or 10.8 mg/day, ip), were compared against hypoxia-induced increases in pulmonary artery pressure and plasma ET concentrations. SB 247083, but not A-192621, inhibited hypoxia-induced pulmonary hypertension, whereas A-192621, but not SB 247083, significantly exacerbated hypoxia-induced increases in ET concentrations, suggesting that hypoxia-induced pulmonary pressor responses are mediated via ETA receptor activation, while ETB receptor blockade may alter clearance of hypoxia-induced elevated plasma ET. The inhibitory effects of SB 217242 on the functional and remodeling changes induced by hypoxia provide further evidence that ET may play a central role in pulmonary hypertension and that ET receptor antagonists may have a utility in the treatment of this disease.

Antiasthmatic activity of the second-generation phosphodiesterase 4 (PDE4) inhibitor SB 207499 (Ariflo) in the guinea pig.

We evaluated the airway activity of the novel phosphodiesterase type 4 inhibitor SB 207499 [Ariflo; c-4-cyano-4-(3-cyclopentyloxy-4-methoxyp henyl-r-1-cyclohexane carboxylic acid)], in the guinea pig. Ovalbumin (OA)-induced contractions of guinea pig isolated tracheal strips were inhibited by SB 207499 with an EC50 of 1 microM but had little or no effect on exogenous agonist-induced contraction, which suggests that its effect on OA-induced contraction in vitro is primarily due to inhibition of mediator release from mast cells. In anesthetized guinea pigs, SB 207499 inhibited OA-induced bronchoconstriction with i.v. and p.o. ID50 values of 1.7 and 17 mg/kg, respectively. At 1, 3 and 6 hr after SB 207499 (30 mg/kg p.o.), OA-induced bronchospasm was inhibited by 92%, 70% and 58%, respectively, corresponding to elevated plasma concentrations of 1.62 +/- 0.19, 1.65 +/- 0.29 and 0. 93 +/- 0.24 microg/ml, respectively, of SB 207499. SB 207499 also inhibited house dust mite-induced bronchoconstriction (ID50 = 0.9 mg/kg i.v. and 8.9 mg/kg p.o.). In contrast to its lack of bronchorelaxant activity in vitro, SB 207499 inhibited bronchospasm induced by i.v. leukotriene D4 (LTD4) [ID50 = 3 mg/kg i.v.]. The bronchorelaxant effect of i.v.-administered SB 207499 was at least additive with that of salbutamol in reversing infused histamine-enhanced airway tone, but it did not alter base line or enhance salbutamol-induced cardiovascular effects. In conscious guinea pigs, SB 207499 (10 or 30 mg/kg p.o.), 1 hr before antigen or LTD4 challenge, markedly reduced bronchospasm and subsequent eosinophil influx as measured by bronchoalveolar lavage 24 hr after provocation. SB 207499 administered after OA or LTD4 challenge also reduced airway eosinophilia measured at 24 hr after OA challenge or 96 hr after LTD4 challenge. These results, coupled with the broad anti-inflammatory activity of SB 207499 previously described (Barnett et al., 1998), suggest that SB 207499 will be useful in the treatment of asthma and other inflammatory disorders.

Food restriction-mediated adrenal influences on antigen-induced bronchoconstriction and airway eosinophil influx in the guinea pig.

The aim of this study was to measure the effects of food restriction on antigen-induced bronchoconstriction and inflammatory cell influx in guinea pigs and to determine the role of plasma cortisol and catecholamine concentrations. Ovalbumin (OA; 0.3 mg/kg, i.v.) was administered to OA-sensitized, anesthetized guinea pigs which had been allowed free access to food or had been food restricted for 18 h prior to OA challenge. In addition to higher plasma levels of epinephrine (30% increase) and cortisol (33% increase), fasted guinea pigs had significantly lower (60% decreased) maximal bronchoconstrictor responses to OA than nonfasted, sensitized litter mates. Additionally, groups of fasted or fed animals were subdivided into two additional treatment groups: (1) saline-pretreated or (2) polyethylene glycol 400 (PEG)-pretreated (1 ml/kg, p.o., 1 h prior to antigen challenge). In saline-treated, fasted animals, bronchoconstrictor responses to antigen were significantly diminished (67% decreased) and epinephrine and cortisol levels were increased (64 and 34%, respectively) compared to the corresponding fed group. In both fasted and fed groups, the PEG-treated guinea pigs had higher plasma epinephrine and cortisol levels than animals which received saline, but no significant differences were detected within the PEG-treated group. Plasma norepinephrine concentrations were lower in all fasted groups. In a separate model in conscious guinea pigs, there were no differences in aerosol OA-induced bronchoconstriction and eosinophil influx between fasted and fed groups. However, compared to the saline pretreatment group, PEG administration reduced the antigen-induced bronchoconstriction and eosinophilia in both fed and fasted guinea pigs. We speculate that the reduced responsiveness to antigen in fasted versus fed animals may result from food-restriction-induced, stress-related release of epinephrine and cortisol from the adrenal glands, thereby suppressing mast cell degranulation or reducing responsiveness to spasmogenic and chemotactic mediators. In addition, the results suggest that oral dosing with 100% PEG may enhance this phenomenon.

Communicating with elders: effects of speaker-listener distance.

As the distance between two communicators increases, weaker sounds of speech become inaudible, conversation becomes more difficult and there is a greater need to resolve communication difficulties. This study investigated the effects of distance on face-to-face social conversation by 27 older adults with hearing impairment, under three listening conditions (without amplification, with personal hearing aids, with a small portable amplifier). Each participant was rated on a 1-5 scale of conversational fluency at distances between 0.5 and 3.0 metres. Results indicate that the subjects conversed more fluently with amplification and at short communication distances. Use of a portable amplifier resulted in high conversational fluency regardless of distance.

Chronic hypoxia-induced cardiopulmonary changes in three rat strains: inhibition by the endothelin receptor antagonist SB 217242.

The cardiopulmonary profile of three different rat strains was compared after exposure to hypoxia (9% O2) for 0, 7, or 14 days. In Sprague-Dawley (SD), Wistar (W), and high altitude-sensitive (HAS) rats, pulmonary arterial pressure (PAP) rose 30, 58, and 85% respectively, after 7 days of hypoxia, and by 108, 116, and 167%, respectively, at 14 days compared to strain- and age-matched normoxic controls. Right ventricular hypertrophy (RVH), expressed as the ratio of right free wall/left wall + septum weight, in SD, W, and HAS was increased by 24, 53, and 48%, respectively, at 7 days, and by 51, 93, and 55% at 14 days compared to normoxic littermates. Histologically, marked medial thickening and luminal stenosis of small and medium-sized arteries were observed in all hypoxic rats, being most pronounced in the HAS rats at 14 days. Treatment of HAS rats with the ET receptor antagonist SB 217242 (3.6 or 10.8 mg/day i.p. by osmotic pump) significantly inhibited the hypoxia-induced increases in PAP (70-75% decrease). RVH was inhibited by 40% at the dose of 10.8 mg/day. Histologically, the SB 217242-treated rats had almost "normal" small and medium-sized arteries, comparable to those of the normoxic HAS controls. This study demonstrates an exaggerated PAP response to chronic hypoxia in HAS compared to SD and W rats. The inhibitory influence of SB 217242 on the functional and morphologic changes induced by hypoxia provides further evidence for a role for ET and the potential utility of ET receptor antagonists in the treatment of pulmonary hypertension.

1,4-Cyclohexanecarboxylates: potent and selective inhibitors of phosophodiesterase 4 for the treatment of asthma.

Evaluation of a variety of PDE4 inhibitors in a series of cellular and in vivo assays suggested a strategy to improve the therapeutic index of PDE4 inhibitors by increasing their selectivity for the ability to inhibit PDE4 catalytic activity versus the ability to compete for high affinity [3H]rolipram-binding sites in the central nervous system. Use of this strategy led ultimately to the identification of cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxyl ic acid (1, SB 207499, Ariflo), a potent second-generation inhibitor of PDE4 with a decreased potential for side effects versus the archetypic first generation inhibitor, (R)-rolipram.

Hypoxia-induced pulmonary hypertension in an optimized environment for the guineapig.

Prolonged exposure to hypoxia elicits a variety of time-related morphologic and physiologic changes in the pulmonary vasculature of mammals, including humans. The study of hypoxia-induced changes in rodents generally requires a prolonged exposure to 9% oxygen for a minimum of 10 days in an airtight chamber, which has only been generally described in the literature as large (200-400 l), sealed acrylic chambers. To assist in the search for better therapies for diseases associated with chronic hypoxia using animal models, we have custom-built an airtight chamber for hypoxic exposure of rodents, and characterized the effect of chronic hypoxia on functional and morphologic changes in the pulmonary vasculature of the guineapig using this system. This chamber has been designed to alleviate any unnecessary stress related to food or water intake, cleanliness and excess illumination to the animals during the hypoxic-exposure period. Chronic exposure of the guineapig to hypoxia (0-21 days) produced time-related physiologic, morphologic, and haematologic changes. For example, after 10 days in hypoxia (9% oxygen), pulmonary artery pressure was significantly increased from 13 +/- 1 mmHg in normoxic controls (day 0, n = 6) to 26 +/- 0 mmHg (day 10, n = 4, P < 0.01). Right ventricular hypertrophy in hypoxic animals, presented as a ratio of right ventricle free wall weight to body weight, showed a significant increase from 0.054 +/- 0.004 (day 0) to 0.069 +/- 0.004 on day 10 (P < 0.05), while age-matched normoxic animals showed no changes in right ventricular weight (day 0 = 0.059, day 10 = 0.058; P > 0.05). Red blood cell count significantly increased over the same time period, from 5.9 +/- 0.1 (day 0) to 6.4 +/- 0.1 (day 10, P < 0.05), as did haematocrit, 48 +/- 0.7 (day 0) to 61 +/- 0.9 (day 10, P < 0.05), and haemoglobin, 16 +/- 0.2 (day 0) to 20 +/- 0.1 (day 10, P < 0.05). It is concluded that considerations for the well-being of the test animals (i.e. continuous water, ample food supplies, burrow-like hiding places, sanitation and protection from excess illumination) can easily be incorporated into a hypoxic chamber. The purpose of the present study was to explore modifications that may provide the animal with an optimized environment which will reduce anxiety and stress, as seen in their behaviour when inside the chambers, and to thoroughly characterize the morphologic and physiologic changes associated with chronic hypoxia which develop in a consistent time-related manner.

The influence of endogenous catecholamines on the inhibitory effects of rolipram against early- and late-phase response to antigen in the guinea pig.

Selective inhibitors of the low-Km cAMP-specific phosphodiesterase (PDE4) inhibit inflammatory cell function and relax airway smooth muscle. Thus PDE4 inhibitors may be useful in the therapy of asthma. The present study was conducted to determine whether the in vivo activity of rolipram, a prototypical PDE4 inhibitor, is due to its ability to potentiate the anti-inflammatory effects of prostaglandins or catecholamines, endogenous activators of adenylyl cyclase, in models of the early- and late-phase response to antigen. Rolipram, administered i.v. to anesthetized, paralyzed and ventilated ovalbumin-sensitized guinea pigs, inhibited i.v. antigen-induced bronchoconstriction with an ID50 value of 0.2 mg/kg. Pretreatment with either of the beta adrenoceptor antagonists propranolol and nadolol (0.5 mg/kg i.v.), enhanced the bronchial reactivity to antigen and abolished the inhibitory activity of rolipram (0.1-10 mg/kg i.v.). In addition, the inhibitory activity of three structurally dissimilar PDE4 inhibitors was nearly abolished by propranolol. Cyclooxygenase inhibition by indomethacin slightly enhanced the reactivity to antigen but did not affect the inhibitory activity of rolipram. Plasma catecholamine concentrations were not altered by rolipram (0.3 or 1 mg/kg i.v.), which indicates that there was no stimulation of catecholamine release. Bilateral adrenalectomy reduced plasma epinephrine concentrations (from 1700 pg/ml to 400 pg/ml), significantly enhanced airway reactivity to antigen and substantially reduced the inhibitory activity of rolipram (3 mg/kg i.v.). Pretreatment of conscious guinea pigs with the beta adrenoceptor antagonist nadolol, 2 mg/kg p.o., enhanced aerosol antigen-induced bronchoconstriction and pulmonary eosinophil influx measured by bronchoalveolar lavage. Nadolol reduced the inhibitory effect of rolipram against antigen-induced bronchoconstriction but not eosinophil influx. The inhibitory effect of rolipram was unaffected by indomethacin. The present data suggest that circulating catecholamines play an important protective role against antigen-induced broncho-constriction in the guinea pig. Moreover, the inhibitory activity of PDE4 inhibitors against antigen-induced bronchoconstriction, but not eosinophil influx, is reduced by beta adrenergic blockade or adrenalectomy. Thus the inhibitory activity of PDE4 inhibitors against antigen-induced bronchoconstriction may be related to their synergism with endogenous catecholamines to suppress mast cell degranulation.

Nonpeptide endothelin receptor antagonists. X. Inhibition of endothelin-1- and hypoxia-induced pulmonary pressor responses in the guinea pig by the endothelin receptor antagonist, SB 217242.

This study investigated the effects of the nonpeptide endothelin (ET) receptor antagonist, SB 217242, against ET-1-induced pulmonary pressor responses and in a model of hypoxia-induced pulmonary hypertension in the guinea pig. In guinea pig isolated pulmonary artery rings, SB 217242 (3-300 nM) produced a concentration-dependent inhibition of ET-1-induced contractions, with a pA2 of 8.1. SB 217242 (1 or 3 mg/kg i.v.) elicited a dose-related inhibition of ET-1-induced increases in pulmonary artery and airway insufflation pressure responses in anesthetized guinea pigs. Chronic exposure to hypoxia (9% O2 for 0-14 days) produced a time-dependent increase in mean pulmonary artery pressure. After a 10-day exposure to hypoxia there was about a 100% elevation in pulmonary artery pressure, and right ventricular mass and plasma irET levels increased 3-fold compared with normoxic animals. SB 217242, administered by continuous intraperitoneal infusion via mini osmotic pump (0.36, 3.6 or 10.8 mg/day), significantly reduced (by about 50%) hypoxia-induced pulmonary artery pressure increases at all three doses used. The hypoxia-induced right ventricular hypertrophy was significantly attenuated by the 3.6 and 10.8 mg/day doses. Based on hematocrit, hemoglobin and red blood cell counts, SB 217242 did not affect the normal physiological erythropoietic response to hypoxia. There were no appreciable differences in the maximum contractile effects of ET-1 or the potency of SB 217242 (pKB values, 8.3 and 8.0, respectively) versus ET-1-induced responses in isolated pulmonary arteries from hypoxic versus normoxic guinea pigs. However, there was a marked reduction in endothelium-dependent relaxation of precontracted pulmonary artery isolated from hypoxic compared with normoxic animals. The results of the present study provide further preclinical evidence for a pathophysiological role of ET-1 and the potential therapeutic utility of ET receptor antagonists, such as SB 217242, in pulmonary hypertension.

Persistent airway eosinophilia after leukotriene (LT) D4 administration in the guinea pig: modulation by the LTD4 receptor antagonist, pranlukast, or an interleukin-5 monoclonal antibody.

Aerosolized cysteinyl leukotrienes (CysLTs) elicit migration of eosinophils into guinea pig lungs and the airways of patients with asthma. The present studies were designed to analyze the concentration-response relationship, time course, and pharmacologic and histologic characteristics of leukotriene D4 (LTD4)-induced eosinophil influx into the airways of conscious guinea pigs. Animals were exposed to aerosols of 0.3 to 30 microg/ml LTD4 for 1 min, during which specific airway conductance (sGaw) was monitored. Bronchoalveolar lavages (BALs) of guinea pig airways were conducted at selected times from 4 h to 4 wk after LTD4 challenge. LTD4 produced maximal decreases in sGaw (70 to 90% reduction) at all concentrations tested and concentration-related increases in eosinophil levels in BALs, assessed 24 h after challenge. Increased numbers of eosinophils in the bronchial epithelium and subepithelium were confirmed histologically. Significant eosinophilia was maintained for up to 4 wk postchallenge. Pretreatment with the LTD4 receptor antagonist, pranlukast (ONO-1078, SB 205312) (20 mg/kg, intragastrically), significantly inhibited both the bronchoconstriction and the eosinophilia at 24 h, whereas the cyclooxygenase inhibitor, meclofenamic acid (5 mg/kg, intragastrically), had no effect on either parameter. Histologic observations were consistent with BAL results. Pretreatment with the rat anti-mouse antibody to interleukin-5 (IL-5), TRFK-5 (10-300 microg, intraperitoneally), produced dose-related inhibition of LTD4-induced eosinophilia, measured in 24 h or 3 wk BAL, but did not affect the acute bronchoconstriction. These results indicate that LTD4 elicits airway eosinophil influx in guinea pigs which persists as long as 4 wk after a single exposure, and provide the first evidence that IL-5 may have a role in LTD4-induced airways inflammation. This and other previously reported proinflammatory effects of LTD4 may contribute significantly to its overall influential role in the pathophysiology of asthma, and may underlie the therapeutic benefit of CysLT receptor antagonists, such as pranlukast, in this disorder.

Catecholamine and beta-adrenoceptor influences on airway reactivity to antigen in guinea pigs.

The aim of the present study was to analyze the increased airway reactivity to antigen induced by beta-adrenoceptor blockade, adrenalectomy or medullectomy and to assess the contribution of circulating catecholamines to the increased reactivity. In anesthetized guinea pigs sensitized to ovalbumin (OA), administration of OA produced a dose-related bronchoconstriction characterized by threshold increases in airway insufflation pressure at 0.1 mg/kg i.v. and a near-maximal increase by 0.3 mg/kg i.v. Pretreatment with R(+) propranolol (0.5 mg/kg i.v.) 5 min prior to antigen did not significantly alter airway responses to antigen when compared to vehicle-treated animals. However, pretreatment with 0.5 mg/kg i.v. S(-) propranolol, racemic propranolol or nadolol markedly enhanced (10- to 15-fold) the airway response to the low-dose antigen. In addition, in guinea pigs which had been adrenalectomized, the reactivity to low-dose antigen was enhanced to a similar extent as that of beta-antagonist-treated animals when compared to sham-operated animals. Baseline plasma concentrations of epinephrine were significantly higher in sham-operate guinea pigs (1,494 +/- 223 ng/ml) when compared to adrenalectomized animals (412 +/- 44 ng/ml). Upon antigen exposure, epinephrine levels rose 5-fold (6,859 +/- 1,308 ng/ml) from baseline in sham-operated guinea pigs and were not significantly changed in adrenalectomized animals (848 +/- 208 ng/ml). Specific airway conductance measurements in conscious guinea pigs revealed that animals which had been medullectomized 2 weeks previously responded to lower provocative concentrations of aerosol OA (0.05-0.5%) than corresponding sham-operated animals. Airway reactivity to inhaled acetylcholine (0.1-1%) was similar in medullectomized and sham guinea pigs. Plasma concentrations of epinephrine were significantly lower in medullectomized guinea pigs (327 +/- 88 ng/ml) when compared to sham-operated animals (832 +/- 162 ng/ml). The results of the present study indicate that beta-adrenoceptor antagonism or changes in circulating epinephrine levels markedly alter the response to antigen in sensitized guinea pigs.

Comparison of phosphodiesterase III, IV and dual III/IV inhibitors on bronchospasm and pulmonary eosinophil influx in guinea pigs.

Selective inhibition of phosphodiesterase (PDE) isozymes has been shown to inhibit inflammatory cell function and relax airway smooth muscle and, thus, may be useful in the therapy of asthma. In guinea pigs sensitized to ovalbumin (OA), the effects of three PDE inhibitors were compared: siguazodan (PDE III selective, IC50 = 0.7 microM), rolipram (PDE IV selective, IC50 = 0.8 microM) and zardaverine (dual PDE III/IV, IC50S = 2.5 microM and 1.1 microM, respectively) against histamine-, leukotriene (LT) D4- and OA-induced bronchospasm in vitro and in vivo. Rolipram or zardaverine (0.1-10 microM), but not siguazodan, inhibited OA-induced contraction of the isolated trachea in a concentration-dependent manner. Rolipram or siguazodan alone (10 microM) were ineffective against histamine- or LTD4-induced contractions. Zardaverine alone (10 microM) or the combination of rolipram and siguazodan (10 microM each) markedly antagonized the contractions elicited by both spasmogens. In anesthesized, ventilated guinea pigs, the i.v. ID50S against OA-induced bronchospasm were: rolipram = 0.2 mg/kg, siguazodan > 10 mg/kg and zardaverine = 2.4 mg/kg. When administered at doses up to 7.5 mg/kg, i.v., rolipram or siguazodan were markedly less effective (i.e., < or = 50% inhibition) than zardaverine (ID50S = 2.4 and 1.7 mg/kg, respectively) at blocking exogenous histamine- or LTD4-induced bronchospasm. However, when administered in combination with siguazodan (5.4 mg/kg, i.v.), rolipram (0.4-5.4 mg/kg) abolished histamine- and LTD4-induced bronchoconstriction. In conscious guinea pigs, zardaverine (5 mg/kg, intragastrically (i.g.) or the combination of rolipram and siguazodan (5 mg/kg each) were substantially more effective than rolipram or siguazodan alone at inhibiting aerosol histamine- or LTD4-induced bronchospasm. In the same animals, rolipram or zardaverine (5 mg/kg, i.g.) but not siguazodan (5 mg/kg, i.g.) markedly inhibited aerosol OA-induced bronchoconstriction. The OA-induced pulmonary eosinophil infiltration in these animals was attenuated by all treatments with zardaverine producing the greatest degree of inhibition. These results indicate that 1) PDE IV inhibitors but not PDE III inhibitors are effective at blocking antigen-induced bronchospasm, 2) compounds that selectively inhibit either PDE III or PDE IV are poor inhibitors of bronchoconstriction elicited by exogenously administered spasmogens, and 3) the combined inhibition of both PDE III and PDE IV isozymes acts in an additive or synergistic manner to inhibit bronchospasm in the guinea pig.

Inhibition of antigen-induced bronchoconstriction and eosinophil infiltration in the guinea pig by the cyclic AMP-specific phosphodiesterase inhibitor, rolipram.

Selective inhibition of the low Km cyclic AMP-specific phosphodiesterase has been shown to inhibit inflammatory cell function and relax airway smooth muscle. These studies were conducted to characterize the bronchodilator and anti-inflammatory activity of rolipram, an archetypical cyclic AMP-specific phosphodiesterase inhibitor, in in vitro and in vivo guinea pig airway models. In isolated tracheal rings from ovalbumin (OA)-sensitive guinea pigs, both R- and S-enantiomers of rolipram (1 microM) significantly antagonized OA-induced contractions. In contrast, neither enantiomer at concentrations up to 1 microM significantly inhibited histamine- or LTD4-induced contractions. In superfusion and mediator release experiments, both enantiomers of rolipram significantly reduced antigen-induced prostaglandin D2 release, but had minimal effect on histamine release. In anesthetized, ventilated OA-sensitive guinea pigs, racemic rolipram or enantiomers reduced OA-induced bronchoconstriction with ID50 values of approximately 0.25 mg/kg i.v. Histamine- and leukotriene D4-induced bronchoconstriction were not affected by doses of rolipram which abolished the response to OA. Higher doses (3-10 mg/kg) reduced histamine-, but not the leukotriene D4-induced bronchoconstriction. In conscious OA-sensitive guinea pigs, intragastric pretreatment with rolipram dose-dependently reduced both the OA-induced decreases in specific conductance as well as the corresponding pulmonary eosinophil influx as assessed by both bronchoalveolar lavage and histological evaluation. Therefore, rolipram produces significant inhibition of antigen-induced bronchoconstrictor and inflammatory responses, thus providing strong evidence that this pharmacological approach may be of significant therapeutic value in allergic asthma.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of late-phase airway responses in conscious guinea pigs after a variety of antigen challenges.

Guinea pigs were actively sensitized by parenteral injections of ovalbumin (OA), house dust extract (HD) or Ascaris suum extract (As) in a variety of multidose regimens. At least 3 weeks after the initial sensitization injection, aerosols of the appropriate antigen were administered to conscious guinea pigs in a double-chamber body plethysmograph. OA elicited the most consistent and intense bronchoconstriction (BC) as measured by decreases in specific airway conductance (sGAW). The airway responses to As were clearly separable into responders and nonresponders. HD produced essentially no BC. However, intense lacrimation and rhinorrhea occurred in all HD-sensitized, but not unsensitized, animals. No late-phase changes in sGAW or increased reactivity to other spasmogens were seen up to 8 h after any antigen challenge. Eosinophil influx of magnitude similar to that measured by 24 h post-antigen bronchoalveolar lavage (BAL) occurred with all the three antigens. Animals which did not bronchoconstrict to As experienced an equal or greater pulmonary eosinophilia as airway responders. The present data with HD and As suggest that acute BC in response to antigen provocation is not a prerequisite for the eventual pulmonary eosinophilia. The lack of late-phase airway reactions in these models raises a doubt in the direct extrapolation to airway responses in allergic human asthma. The acute lacrimation and rhinorrhea to HD may suggest utility as a model of allergic rhinitis.

SK&F 104353, a selective leukotriene receptor antagonist, inhibits leukotriene D4- and antigen-induced bronchoconstriction in cynomolgus monkeys.

The ability of SK&F 104353 to prevent and reverse leukotriene (LT) D4- and antigen (Ag)-induced bronchoconstriction was examined in anesthetized, spontaneously breathing cynomolgus monkeys. Aerosol administration of LTD4 (10 micrograms/ml; 20 breaths) produced a sustained increase in pulmonary resistance and decrease in dynamic lung compliance. Aerosolized SK&F 104353 (150 breaths, 0.3 or 4.4 mg/ml) administered 15 min prior to LTD4 challenge antagonized these changes in a dose-dependent manner. When given intravenously 6 min after LTD4, SK&F 104353 (5 mg/kg) rapidly and completely reversed the ongoing bronchoconstriction. In mepyramine-pretreated (2 mg/kg i.v.) monkeys sensitive to aerosolized Ascaris suum Ag, intravenously administered SK&F 104353 (5 mg/kg) substantially reversed, but did not abolish, Ag-induced bronchoconstriction when administered 12 min after the Ag challenge. In contrast, SK&F 104353 (5 mg/kg i.v.) did not reverse Ag-induced bronchoconstriction in animals that had not been pretreated with mepyramine. Similar results were obtained when SK&F 104353 (20 mg/kg i.v.) was administered (as a pretreatment) 5 min prior to Ag under these conditions. Thus, SK&F 104353 reduced Ag-induced bronchoconstriction in mepyramine-pretreated monkeys, but had little effect in the absence of mepyramine. The data suggest that LTs, in addition to histamine, play a role in allergic bronchoconstriction in cynomolgus monkeys.