Beta(2)-agonists block tussive responses in guinea pigs via an atypical cAMP-dependent pathway.

beta(2)-Adrenoceptor agonists are the most effective bronchodilators currently available, and are used for symptom management in asthmatics. However, whether beta(2)-agonists are also antitussive is controversial. Identifying an antitussive role for beta(2)-agonists and dissecting the possible mechanism of action may help to explain the inconsistencies in the clinical literature and lead to the development of novel therapeutic agents. The aim of the present study was to determine whether or not beta(2)-agonists attenuate the tussive response in guinea pig and human models, and, if so, to identify the mechanism(s) involved. Depolarisation of vagal sensory nerves (human and guinea pig) was assessed as an indicator of sensory nerve activity. Cough was measured in a conscious guinea pig model. A beta(2)-agonist, terbutaline, dose-dependently inhibited the cough response to tussive agents in conscious guinea pigs. Terbutaline and another beta(2)-agonist, fenoterol, blocked sensory nerve activation in vitro. Using these mechanistic models, it was established that beta(2)-agonists suppress the tussive response via a nonclassical cyclic adenosine monosphosphate-dependent pathway that involves the activation of protein kinase G and, subsequently, the opening of large-conductance calcium-activated potassium channels. In conclusion, beta(2)-adrenoceptor agonists are antitussive, and this property occurs due to a direct inhibition of sensory nerve activation. These findings may help to explain the confusion that exists in the clinical literature, and could be exploited to identify novel therapies for the treatment of cough, which is a significant unmet medical need.

Cough sensors. III. Opioid and cannabinoid receptors on vagal sensory nerves.

Cough is a persistent symptom of many inflammatory airways' diseases. Cough is mediated by receptors sited on sensory nerves and then through vagal afferent pathways, which terminate in the brainstem respiratory centre. Cough is often described as an unmet clinical need. Opioids are the only prescription-based antitussives currently available in the UK. They possess limited efficacy and exhibit serious unwanted side effects, such as physical dependence, sedation, respiratory depression and gastrointestinal symptoms. There are three classical opioid receptors: the mu, kappa and delta receptors. Peripheral opioid receptors are sited on sensory nerves innervating the airways. A greater understanding of the role of the peripheral and centrally sited opioid receptors is necessary to allow the development of targeted treatments for cough. Because of the limited efficacy and the side-effect profile of the opioids, potential new treatments are sought to alleviate cough. One class of compounds that is currently under examination is the cannabinoids. Like the opioids, cannabinoids have peripheral and centrally sited receptors and also suffer from the blight of unwanted centrally mediated side effects such as sedation, cognitive dysfunction, tachycardia and psychotropic effects. Two cannabinoid receptors have been identified, the CB(1) and CB(2) receptors, and their distribution varies throughout the peripheral and central nervous system. Encouragingly, early studies with these compounds suggest that it may be possible to separate their antitussive activity from their centrally mediated side effects, with CB(2) agonists showing potential as putative new treatments for cough. In this chapter, we describe the opioid and cannabinoid receptors, their distribution and the effects they mediate. Moreover, we highlight their potential advantages and disadvantages in the treatment of cough.

Inhibitory activity of the novel CB2 receptor agonist, GW833972A, on guinea-pig and human sensory nerve function in the airways.

BACKGROUND AND PURPOSE: Sensory nerves regulate central and local reflexes such as airway plasma protein leakage, bronchoconstriction and cough. Sensory nerve activity may be enhanced during inflammation such that these protective effects become exacerbated and deleterious. Cannabinoids are known to inhibit airway sensory nerve function. However, there is still controversy surrounding which receptor is involved in eliciting these effects. EXPERIMENTAL APPROACH: We have adopted a pharmacological approach, including using a novel, more selective CB(2) receptor agonist, GW 833972A (1000-fold selective CB(2)/CB(1)), and receptor selective antagonists to investigate the inhibitory activity of cannabinoids on sensory nerve activity in vitro and in vivo in guinea-pig models of cough and plasma extravasation. KEY RESULTS: GW 833972A inhibited capsaicin-induced depolarization of the human and guinea-pig and prostaglandin E(2) (PGE(2)) and hypertonic saline-induced depolarization of the guinea-pig isolated vagus nerve in vitro. GW 833972A also inhibited citric acid-induced cough but not plasma extravasation in the guinea-pig and this effect was blocked by a CB(2) receptor antagonist. CONCLUSIONS AND IMPLICATIONS: This confirms and extends previous studies highlighting the role of CB(2) receptors in the modulation of sensory nerve activity elicited both by the exogenous ligands capsaicin and hypertonic saline but also by endogenous modulators such as PGE(2) and low pH stimuli. These data establish the CB(2) receptor as an interesting target for the treatment of chronic cough.

The heterotopic tracheal allograft as an animal model of obliterative bronchiolitis.

Heterotopic tracheal allografts in small rodents have been shown to share many characteristics with the development of obliterative bronchiolitis (OB) in the clinic and therefore provide a suitable animal model for the study of OB. The model facilitates the examination of the pathogenesis of the disease and the elucidation of the cellular and molecular mechanisms involved in its development. The model provides a less technically demanding alternative to whole lung transplantation in small rodents and should lead to a speedier identification of new treatments that might prevent the development of post-transplantation OB in the clinic.

Evidence that the anti-spasmogenic effect of the beta-adrenoceptor agonist, isoprenaline, on guinea-pig trachealis is not mediated by cyclic AMP-dependent protein kinase.

1. The spasmolytic and anti-spasmogenic activity of beta-adrenoceptor agonists on airways smooth muscle is thought to involve activation of the cyclic AMP/cyclic AMP-dependent protein kinase (PKA) cascade. Here we have tested the hypothesis that PKA mediates the anti-spasmogenic activity of isoprenaline and other cyclic AMP-elevating agents in guinea-pig isolated trachea by utilizing a number of cell permeant cyclic AMP analogues that act as competitive 'antagonists' of PKA. 2. Anion-exchange chromatography of guinea-pig tracheae resolved two peaks of PKA activity that corresponded to the type I ( approximately 5%) and type II ( approximately 93%) isoenzymes. 3. Pre-treatment of tracheae with zardaverine (30 microM), vasoactive intestinal peptide (VIP) (1 microM) and the non-selective activator of PKA, Sp-8-CPT-cAMPS (10 microM), produced a non-parallel rightwards shift in the concentration-response curves that described acetylcholine (ACh)-induced tension generation. The type II-selective PKA inhibitor, Rp-8-CPT-cAMPS (300 microM), abolished this effect. 4. Pre-treatment of tracheae with Sp-8-Br-PET-cGMPS (30 microM) produced a non-parallel rightwards shift of the concentration-response curves that described ACh-induced tension generation. The selective cyclic GMP-dependent protein kinase (PKG) inhibitor, Rp-8-pCPT-cGMPS (300 microM), abolished this effect. 5. Pre-treatment of tracheae with isoprenaline (1 microM) produced a 10 fold shift to the right of the ACh concentration-response curve by a mechanism that was unaffected by Rp-8-Br-cAMPS (300 microM, selective inhibitor of type I PKA), Rp-8-CPT-cAMPS (300 microM) and Rp-8-pCPT-cGMPS (300 microM). 6. We conclude that the anti-spasmogenic activity of Sp-8-CPT-cAMPS, zardaverine and VIP in guinea-pig trachea is attributable to activation of the cyclic AMP/PKA cascade whereas isoprenaline suppresses ACh-induced contractions by a mechanism(s) that is independent of PKA and PKG.

The discovery of RPR 200765A, a p38 MAP kinase inhibitor displaying a good oral anti-arthritic efficacy.

RPR132331, a 2-(2-dioxanyl)imidazole, was identified as an inhibitor of tumour necrosis factor (TNF)alpha release from lipopolysaccharide (LPS)-stimulated human monocytes. An intensive programme of work exploring the biology, toxicity and physical chemistry of a novel series of inhibitors, derived from RPR132331, has led to the identification of RPR200765A, a development candidate for the treatment of rheumatoid arthritis (RA). RPR200765A is a potent and selective inhibitor of p38 MAP kinase (IC50 = 50 nM). It inhibits LPS-stimulated TNFalpha release both in vitro, from human monocytes (EC50 = 110 nM), and in vivo in Balb/c mice (ED50 = 6 mg/kg). At oral doses between 10 and 30 mg/kg/day it reduces the incidence and progression in the rat streptococcal cell wall (SCW) arthritis model when administered in either prophylactic or therapeutic dosing regimens. The compound, which is a mesylate salt and exists as a stable monohydrate, shows good oral bioavailabiltiy (F = 50% in the rat) and excellent chemical stability. The data from the SCW disease model suggests that RPR200765A could exhibit a profile of disease modifying activity in rheumatoid arthritis (RA) patients which is not observed with current drug therapies.

Mediator involvement in antigen-induced bronchospasm and microvascular leakage in the airways of ovalbumin sensitized Brown Norway rats.

1. To determine which mediators are involved in antigen-induced bronchospasm and microvascular leakage in the airways of ovalbumin sensitised Brown Norway rats we investigated the effect of a histamine H(1) receptor antagonist, mepyramine, a 5-HT receptor antagonist, methysergide, and a cys-leukotriene-1 receptor antagonist, montelukast. 2. Ovalbumin at 1 mg kg(-1) i.v. caused a significant increase in microvascular leakage in the airways and at 3 mg kg(-1) i.v. caused a significant increase in airways resistance. 3. Histamine (1 mg kg(-1) i.v.), 5-HT (0.1 mg kg(-1) i.v.) and leukotriene D(4) (LTD(4), 50 microg kg(-1) i.v.) caused a significant increase in microvascular leakage in the airways. 4. Mepyramine (1 mg kg(-1) i.v.), methysergide (0.1 mg kg(-1) i.v.), or montelukast (30 mg kg(-1) i.v.) inhibited histamine, 5-HT or LTD(4) -induced microvascular leakage respectively. 5. Methysergide (0.1 mg kg(-1) i.v.) reduced ovalbumin-induced microvascular leakage in the trachea and at 0.3 mg kg(-1) i.v. inhibited bronchospasm (38 and 58%, respectively). Montelukast (30 mg kg(-1) p.o.) reduced ovalbumin-induced microvascular leakage in airway tissue to basal levels (78%) and inhibited ovalbumin-induced bronchospasm (50%). Mepyramine (3 mg kg(-1) i.v.) had no effect on ovalbumin-induced leakage or bronchospasm. 6. A combination of all three compounds (mepyramine, methysergide and montelukast) reduced ovalbumin-induced microvascular leakage in airway tissue to basal levels (70 - 78%) and almost completely inhibited bronchospasm (92%). 7. Antigen-induced bronchospasm appears to equally involve the activation of 5-HT and cys-leukotriene-1 receptors whereas ovalbumin-induced microvascular leakage appears to be predominantly mediated by cys-leukotriene-1 receptors.

Effect of endothelin antagonists, including the novel ET(A) receptor antagonist LBL 031, on endothelin-1 and lipopolysaccharide-induced microvascular leakage in rat airways.

1. The effect of the novel ET(A) receptor antagonist LBL 031 and other selective and mixed endothelin receptor antagonists on endothelin-1 (ET-1)-induced and lipopolysaccharide (LPS)-induced microvascular leakage was assessed in rat airways. 2. Intravenously administered ET-1 (1 nmole kg(-1)) or LPS (30 mg kg(-1)) caused a significant increase in microvascular leakage in rat airways when compared to vehicle treated animals. 3. Pre-treatment with the selective ET(A) receptor antagonists, LBL 031 or PD 156707, or the mixed ET(A/B) receptor antagonist, bosentan (each at 30 mg kg(-1)), reduced ET-1-induced leakage to baseline levels. ET-1-induced leakage was not reduced by pre-treatment with the ET(B) selective antagonist BQ 788 (3 mg kg(-1)). 4. Pre-treatment with the selective ET(A) receptor antagonist, LBL 031 (0.1 mg kg(-1)) or PD 156707 (10 mg kg(-1)), or the mixed ET(A/B) receptor antagonist, bosentan (30 mg kg(-1)), reduced LPS-induced leakage by 54, 48 and 59% respectively. LPS-induced leakage was not affected by pre-treatment with the ET(B) selective antagonist BQ 788 (3 mg kg(-1)). 5. The data suggests that ET-1-induced microvascular leakage in the rat airway is ET(A) receptor mediated and that part of the increase induced by LPS may be due to the actions of ET-1. Therefore, a potent ET(A) receptor selective antagonist, such as LBL 031, may provide a suitable treatment for inflammatory diseases of the airways, especially those involving LPS and having an exudative phase, such as the septic shock-induced adult respiratory distress syndrome.

Effect of the p38 kinase inhibitor, SB 203580, on sephadex induced airway inflammation in the rat.

SB 203580 is a pyridinyl imidazole compound which inhibits the release of pro-inflammatory cytokines, such as tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), in vitro and in vivo by inhibiting p38 mitogen-activated protein kinase (MAPK). The present study investigated the effects of SB 203580 in a model of airway inflammation induced by the topical administration of Sephadex into the rat airways. This inflammatory response is characterized by the development of lung oedema, airway tissue inflammatory cell recruitment and an increase in lung TNF-alpha and IL-1beta levels. Sephadex-induced lung oedema was accompanied by a significant increase in lung tissue TNF-alpha but not IL-1beta levels. There was also a significant increase in lung tissue macrophages and an increase in eosinophils which did not reach significance. SB 203580 administration significantly inhibited lung oedema (ED50=18 mg x kg(-1)) in a dose-related manner but was without significant effect on lung tissue cell recruitment or cytokine levels. These data suggest that the increase in tumour necrosis factor-alpha and lung oedema are separate processes which both contribute to Sephadex pathology. Furthermore, the inhibitory effect of SB 203580 on Sephadex-induced lung oedema suggests that p38 kinase inhibitors may be of use in pulmonary pathologies in which lung oedema is a feature.

RPR 107393, a potent squalene synthase inhibitor and orally effective cholesterol-lowering agent: comparison with inhibitors of HMG-CoA reductase.

Squalene synthase catalyzes the reductive dimerization of two molecules of farnesyl pyrophosphate to form squalene and is the first committed step in sterol synthesis. A specific inhibitor of squalene synthase would inhibit cholesterol biosynthesis but not prevent the formation of other products of the isoprenoid pathway, such as dolichol and ubiquinone. RPR 107393 [3-hydroxy-3-[4-(quinolin-6-yl)phenyl]-1-azabicyclo[2-2-2]octane dihydrochloride] and its R and S enantiomers are potent inhibitors of rat liver microsomal squalene synthase, with IC50 values of 0.6 to 0.9 nM. One hour after oral administration to rats, RPR 107393 inhibited de novo [14C]cholesterol biosynthesis from [14C]mevalonate in the liver with an ED50 value of 5 mg/kg. Diacid metabolites of [14C]farnesyl pyrophosphate were identified after acid treatment of the livers of these animals. These results support in vitro data demonstrating that these compounds are inhibitors of squalene synthase. In rats, RPR 107393 (30 mg/kg p.o. b.i.d. for 2 days) reduced total serum cholesterol by < or = 51%. In the same paradigm, the HMG-CoA reductase inhibitor lovastatin failed to lower serum cholesterol in rats. In marmosets, RPR 107393 (20 mg/kg b.i.d.) reduced plasma cholesterol concentration by 50% after 1 week of administration; this was greater than the reduction observed with lovastatin or pravastatin, neither of which produced > 31% reduction in plasma cholesterol when administered for 1 week at a dose of 50 mg/kg b.i.d. The R and S enantiomers of RPR 107393 (20 mg/kg p.o. q.d. for 7 days) reduced plasma low density lipoprotein cholesterol by 50% and 43%, respectively, whereas high density lipoprotein cholesterol was unchanged. In summary, RPR 107393 is a potent inhibitor of squalene synthase. It is an orally effective hypocholesterolemic agent in rats and marmosets that has greater efficacy than lovastatin or pravastatin in the marmoset.

Hypolipidaemic properties of a potent and bioavailable alkylsulphinyl-diphenylimidazole ACAT inhibitor (RP 73163) in animals fed diets low in cholesterol.

RP 73163 ((S)-2-[5-(3,5-dimethyl-l-pyrazolyl)pent-l-yl)-sulphinyl]-5, 6-diphenylimidazole) has been shown to be a potent and specific inhibitor of acyl-CoA:cholesterol acyltransferase (EC 2.3.1.26; ACAT) in vitro using the tissues of experimental animals as sources of the enzyme. The concentrations of RP 73163 required to produce 50% inhibition of ACAT activity (IC50 values) in microsomal preparations ranged from 86 nM for rat liver to 370 nM for rabbit intestine. In whole cell assays using human hepatic (HepG2), intestinal (Caco2), and monocytic (THP-1) cell lines, RP 73163 inhibited ACAT activity with IC50 values of 266, 158, and 314 nM, respectively. The addition of RP 73163 (0.03-1.0 microM) to the medium of cultured HepG2 cells produced a concentration-dependent decrease in apolipoprotein B (apoB) secretion. The compound has high systemic bioavailability. Using a bioassay, a concentration of active inhibitor equivalent to 29 microM of parent compound was present in plasma 1 hr after oral administration of RP 73163 (50 mg.kg-1). In rats that had been fed a basal diet ad libitum or starved for 18 hr prior to blood sampling, the administration of RP 73163 (50 mg.kg-1 b.i.d. for 7 days) reduced plasma triglyceride levels by 50% without affecting the concentration of cholesterol. This hypotriglyceridaemic effect was associated with reductions in plasma very-low-density-lipoprotein (VLDL) and low-density-lipoprotein (LDL) levels. RP 73163 decreased the rate of VLDL secretion by 24% in Triton WR-1339-treated rats that had been fasted overnight but did not affect the secretion rate in animals fed ad libitum, indicating that ACAT was only important in regulating VLDL secretion under certain nutritional conditions. RP 73163 reduced the accumulation of intraperitoneally administered [3H]leucine into the plasma VLDL-apoB pool in both fed and fasted states. The results suggest that, in fed animals at least, an increase in the clearance of VLDL from the bloodstream may contribute to the hypolipidaemic activity of the compound. In rabbits with casein-induced endogenous hypercholesterolaemia, RP 73163 specifically reduced the levels of cholesterol carried by LDL. In conclusion, the hypolipidaemic actions of RP 73163, a potent and systemically bioavailable ACAT inhibitor, are consistent with a reduction in the secretion of apoB containing lipoproteins by hepatic tissue and possibly with an increase in the clearance of these particles.

RPR 101821, a new potent cholesterol-lowering agent: inhibition of squalene synthase and 7-dehydrocholesterol reductase.

RPR 101821 (trans-2-[4-(benzoxazol-2-yl)-phenylmethoxy] amino cyclohexane hydrochloride) is a potent cholesterol-lowering agent in rodents and marmoset. The compound inhibited rat liver microsomal squalene synthase (IC50 = 1 nM) and 7-dehydrocholesterol (7DHC) reductase (IC50 = 1 microM; Lewis et al. 1995). When RPR 101821 (10 mg/kg), the 7DHC reductase inhibitor BM 15.766 (4[2-[4-(4-chlorocinnamyl)piperazine-1-yl]ethyl] benzoic acid; 10 mg/kg) or the HMG-CoA reductase inhibitor lovastatin (30 mg/kg) was given orally to rats at -29 h, -21 h and -5 h, serum cholesterol was reduced by 56%, 46% or 15%, respectively. The reduction in cholesterol with RPR 101821 was associated with an accumulation of 7DHC in serum, suggesting an inhibition of 7DHC reductase. In the presence of BM 15.766, RPR 101821 reduced the serum accumulation of 7DHC in a dose-dependent manner, with complete inhibition at 30 mg/kg, p.o. In Balb-cJ mice, RPR 101821 and lovastatin (50 mg/kg, b.i.d., p.o., for 14 days) lowered serum cholesterol by 67% and 2%, respectively. In marmosets, RPR 101821 and lovastatin (both at a dose of 10 mg/kg, p.o., b.i.d., for 7 days) reduced cholesterol by 28% and 19%, respectively. In summary, RPR 101821 is an orally effective potent cholesterol-lowering agent in rodents and a small primate species. The suggested mechanism of hypocholesterolemic effect is the inhibition of squalene synthase and 7DHC reductase.

Measurement of the absolute number of functioning low-density lipoprotein receptors in vivo using a monoclonal antibody.

MAC188 S/S is a monoclonal antibody which can be used in vivo to measure the absolute number of functioning low-density lipoprotein (LDL) receptors in a rabbit. The antibody binds to the extra-cellular domain of the LDL receptor and binding is not blocked by the presence of LDL. When the antibody-receptor complex is internalized, receptor recycling is inhibited for several hours. Thus when saturating doses of MAC188 S/S are administered intravenously, the amount of antibody removed from the blood (minus non-specific removal) is determined solely by the total number of LDL receptors in an animal. In this study MAC188 S/S was used to measure the number of LDL receptors in control rabbits and in animals treated with 17 alpha-ethinyl oestradiol. After treatment (which caused a 47% decrease in plasma cholesterol), receptor-mediated removal of MAC188 S/S from the blood was saturated in both groups following injection of 3.0 mg of antibody per kg body weight. Based on the amount of antibody removed via the LDL receptor at this dose, the total number of accessible LDL receptors was calculated as (2.0 +/- 0.3) x 10(15) receptors per kg body weight in control rabbits and (4.0 +/- 0.4) x 10(15) receptors per kg body weight in oestrogen-treated animals. The number of receptors in various organs was also determined. The monoclonal antibody approach therefore, allows accurate determination of LDL receptor numbers in animals with markedly different concentrations of circulating LDL, conditions in which the use of endogenous ligand would be subject to significant errors.