Dual bronchodilatory and pulmonary anti-inflammatory activity of RO5024118, a novel agonist at vasoactive intestinal peptide VPAC2 receptors.

BACKGROUND AND PURPOSE: Vasoactive intestinal peptide is expressed in the respiratory tract and induces its effects via its receptors, VPAC(1) and VPAC(2). RO5024118 is a selective VPAC(2) receptor agonist derived via chemical modification of an earlier VPAC(2) agonist, RO0251553. In the present studies, we characterized the pharmacological activity of RO5024118. EXPERIMENTAL APPROACH: Stability of RO5024118 to human neutrophil elastase was assessed. Bronchodilatory activity of RO5024118 was investigated in guinea pig and human isolated airway smooth muscle preparations and in a guinea pig bronchoconstriction model. Pulmonary anti-inflammatory activity of RO5024118 was investigated in a lipopolysaccharide mouse model and in a porcine pancreatic elastase (PPE) rat model. KEY RESULTS: RO5024118 demonstrated increased stability to neutrophil elastase compared with RO0251553. In human and guinea pig isolated airway preparations, RO5024118 induced bronchodilatory effects comparable with RO0251553 and the long-acting ß-agonist salmeterol and was significantly more potent than native vasoactive intestinal peptide and the short-acting ß-agonist salbutamol. In 5-HT-induced bronchoconstriction in guinea pigs, RO5024118 exhibited inhibitory activity with similar efficacy as, and longer duration than, RO0251553. In a lipopolysaccharide-mouse model, RO5024118 inhibited neutrophil and CD8(+) cells and myeloperoxidase levels. In rats, intratracheal instillation of PPE induced airway neutrophilia that was resistant to dexamethasone. Pretreatment with RO5024118 significantly inhibited PPE-induced neutrophil accumulation. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that RO5024118 induces dual bronchodilatory and pulmonary anti-inflammatory activity and may be beneficial in treating airway obstructive and inflammatory diseases. LINKED ARTICLES: This article is part of a themed section on Analytical Receptor Pharmacology in Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2010.161.issue-6.

The interleukin-12/interleukin-12-receptor system: role in normal and pathologic immune responses.

Interleukin-12 (IL-12) is a heterodimeric cytokine that plays a central role in promoting type 1 T helper cell (Th1) responses and, hence, cell-mediated immunity. Its activities are mediated through a high-affinity receptor composed of two subunits, designated beta 1 and beta 2. Of these two subunits, beta 2 is more restricted in its distribution, and regulation of its expression is likely a central mechanism by which IL-12 responsiveness is controlled. Studies with neutralizing anti-IL-12 antibodies and IL-12-deficient mice have suggested that endogenous IL-12 plays an important role in the normal host defense against infection by a variety of intracellular pathogens. However, IL-12 appears also to play a central role in the genesis of some forms of immunopathology. Inhibition of IL-12 synthesis or activity may be beneficial in diseases associated with pathologic Th1 responses, such as multiple sclerosis or Crohn's disease. On the other hand, administration of recombinant IL-12 may have utility in the treatment of diseases associated with pathologic Th2 responses such as allergic disorders and asthma.

Ro 45-2081, a TNF receptor fusion protein, prevents inflammatory responses in the airways.

The TNF receptor fusion protein, Ro 45-2081, inhibited allergic and non-allergic inflammatory responses in the airways. Treatment of sensitized guinea-pigs with Ro 45-2081 reduced allergen-induced influx of inflammatory cells into the lungs, abolished edema formation and inhibited hyperreactivity to substance P. Administration of Ro 45-2081 after allergen challenge reversed the influx of inflammatory cells into the lungs. Sephadex-induced neutrophil influx into the lungs of rats was also blocked by Ro 45-2081. The effects of Ro 45-2081 suggest that inhibitors of TNF may have potential as therapeutics for inflammatory diseases in the lung.

Pharmacological evidence for tumor necrosis factor as a mediator of allergic inflammation in the airways.

Tumor necrosis factor (TNF) has been implicated in the pathophysiology of a number of inflammatory diseases of the lung. Using the TNF receptor fusion protein, Ro 45-2081, our study investigated the involvement of TNF in allergic inflammatory responses in the airways of sensitized guinea pigs and Brown-Norway rats. Sensitized guinea pigs exhibited an enhanced airway reactivity to substance P (1-10 micrograms/kg, i.v.) at 6 hr after antigen challenge which was inhibited (P < .05) by Ro 45-2081 (3 mg/kg, i.p.). Treatment with Ro 45-2081 (1-3 mg/kg, i.p.) dose-dependently inhibited (P < .05) the accumulation of neutrophils and total cells in bronchoalveolar lavage fluid in sensitized guinea pigs examined at 6 and 24 hr postchallenge. Ro 45-2081 (3 mg/kg, i.p.) also significantly (P < .05) reduced the number of eosinophils in bronchoalveolar lavage at both time points whereas a lower dose (1 mg/kg, i.p.) had no effect. Ro 45-2081 (1 or 3 mg/kg, i.p.) abolished antigen-induced microvascular leakage (quantified by tissue content of Evans blue dye) in the trachea and main bronchi in sensitized guinea pigs. In the Brown-Norway rat, Ro 45-2081 (1-3 mg/kg, i.p.) caused a dose-dependent inhibition of neutrophil and eosinophil infiltration into bronchoalveolar lavage fluid at 24 hr after antigen challenge. In both guinea pig and Brown-Norway rat models, treatment with dexamethasone (30 mg/kg, i.p., for guinea pig and 0.3 mg/kg, i.p., for Brown-Norway rat) produced virtually identical results to those obtained with Ro 45-2081. The ability of Ro 45-2081 to inhibit antigen-induced responses in sensitized animals suggests that TNF is a mediator of allergic inflammation in the lung.

Inhibition of Sephadex-induced lung injury in the rat by Ro 45-2081, a tumor necrosis factor receptor fusion protein.

Tumor necrosis factor (TNF) is an inflammatory cytokine produced by many cell types which may contribute to the pathophysiology of a variety of lung diseases. In this study we have used Ro 45-2081 (a soluble receptor composed of the human p55 TNF receptor and human heavy-chain immunoglobulin G) to explore the role of TNF in the acute inflammatory response in the rat lung to intravenous injection of Sephadex beads. The effects of Ro 45-2081 have also been compared with those of dexamethasone. At 24 and 72 h after Sephadex, there was a significant increase in the total number of leukocytes in bronchoalveolar lavage fluid (BALF). At 24 h, the number of neutrophils comprised around 50% of the total leukocyte number, decreasing to around 10% of total by 72 h. The eosinophil count was maintained at around 10% of the total leukocyte number. Pretreatment with either Ro 45-2081 [1 and 3 mg kg-1, intraperitoneally (i.p.)] or dexamethasone (0.1 and 0.3 mg kg-1, i.p.) inhibited the neutrophilia at 24 h after Sephadex, although Ro 45-2081 had no significant effect on total cell number. At 72 h after Sephadex, Ro 45-2081 (1 and 3 mg kg-1, i.p., daily) significantly reduced the neutrophil influx into BALF but had no inhibitory effect on eosinophil number. In contrast, dexamethasone (0.1 and 0.3 mg kg-1, i.p., daily) virtually abolished the infiltration of neutrophils and eosinophils into BALF. The lack of effect of Ro 45-2081 on eosinophil infiltration into the rat lung and the inhibition caused by dexamethasone suggest that factors other than TNF are involved in this part of the inflammatory response induced by Sephadex. However, the inhibitory effects of Ro 45-2081 show that TNF may play an important role in the recruitment of neutrophils into the lungs of Sephadex-treated rats.

Effects of Ro 47-0203 on endothelin-1 and sarafotoxin S6c-induced contractions of human bronchus and guinea-pig trachea.

Endothelin exerts a variety of biological effects in the lung which indicate that this peptide may have a role in the pathophysiology of a number of pulmonary diseases. In this study, the endothelin receptors on the human bronchus were compared with those on the guinea-pig trachea using the novel, non-peptide antagonist Ro 47-0203 (4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-2,2' -bipyrimidin-4-yl]-benzenesulphonamide, non-selective for endothelin ETA over endothelin ETB receptor) and the peptide antagonist BQ123 (cyclo(-D-Val-Leu-D-Trp-D-Asp-Pro), endothelin ETA receptor selective). On the human bronchus and guinea-pig trachea, the concentration-effect curve for endothelin-1 was shifted to the right by Ro 47-0203 (100 microM) with concentration ratios of 28.2 +/- 6.8 and 39.5 +/- 13.9, respectively but lower concentrations of the antagonist had no effect. Although the concentration-effect curve for sarafotoxin S6c on the human bronchus was shifted to the right by Ro 47-0203 (30 and 100 microM, concentration ratio: 6.88 +/- 1.72 and 69.7 +/- 17.2, respectively), equivalent degrees of inhibition could be obtained on guinea-pig trachea with lower concentrations of antagonist (10 and 30 microM, concentration ratio: 6.90 +/- 1.58 and 75.8 +/- 14.1 respectively). The lack of effect of BQ123 (10 microM) and the high concentrations of Ro 47-0203 needed to show antagonism indicate that endothelin receptors on both tissues are probably the endothelin ETB subtype. Although the antagonism by Ro 47-0203 is not classically competitive, the greater effect of Ro 47-0203 against sarafotoxin S6c on the guinea-pig trachea may reflect a difference between the endothelin ETB receptors on these tissues.

Modulation of cat antral slow waves by ion substitution, Ca2+ and K+ channel blockade, and ACh stimulation.

Two distinct slow waves can be recorded from smooth muscle cells on the submucosal border (submucosal cells) and the myenteric border (myenteric cells) in the circular layer of the cat antrum. The present studies examined the effects of decreasing extracellular Na+ ([Na+]o) or Ca2+ concentration ([Ca2+]o), Ca2+ and K+ channel blockade, and acetylcholine (ACh) stimulation on "submucosal" and "myenteric" slow waves using intracellular recording techniques. Decreasing [Na+]o (30 mM) reduced the upstroke depolarization of slow waves from submucosal cells but had no effect on the upstroke depolarization of myenteric cells. Decreasing [Ca2+]o (0.5 mM) decreased the upstroke depolarization in cells from each area. Neither cadmium (200 microM) nor nifedipine (100 microM) had any effect on the upstroke depolarizations from both submucosal and myenteric slow waves. In contrast, nickel (5 mM), a nonselective Ca2+ channel blocker, decreased the upstroke depolarizations of slow waves from submucosal and myenteric cells. Slow waves from myenteric cells normally exhibit a plateau phase of several seconds duration. The amplitude of the plateau potential could be decreased by lowering [Na+]o or [Ca2+]o or by nickel, cadmium or nifedipine. In the presence of 30 mM Na+ slow waves from submucosal cells appeared to develop plateau potentials. K+ channel blockade with tetrabutylammonium (TBA, 5 mM) or cesium (10 mM) increased the upstroke depolarization and delayed membrane repolarization in slow waves from submucosal cells. TBA and cesium also increased the upstroke depolarization from myenteric cells but did not delay membrane repolarization. ACh (1 and 3 microM) caused the generation of plateau potentials in slow waves from submucosal cells and increased slow-wave duration.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonadrenergic, noncholinergic contractile responses of the guinea pig hilar bronchus involve the preferential activation of tachykinin neurokinin2 receptors.

Experiments were designed to characterize receptor(s) that mediate nonadrenergic, noncholinergic contractions of the guinea pig hilar bronchus using selective neurokinin (NK)1 (CP 96,345) and NK2 (R396 and MEN 10,376) tachykinin receptor antagonists. Left and right hilar bronchi were studied as pairs in the presence of atropine, propranolol, phentolamine; indomethacin and thiorphan. (2S, 3S)-cis-2-(diphenylmethyl)-N-(2-methoxyphe nyl)-1-azabicyclo[2,2,2]octan-3-amine (CP 96,345) selectively antagonized contractions of the bronchus to the NK1 agonist Ac-[Arg6,Sar9,Met(O2)11]-SP(6-11) with a -log molar KB value of about 8.0. Similarly, Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH2 (R396) and [Tyr5, D-Trp6,8,9, Lys10]-NKA(4-10) (MEN 10,376) selectively antagonized contractions to the NK2 agonist [beta Ala8]-NKA(4-10) with -log molar KB values of about 5.5 and 6.7, respectively. CP 96,345 (3 x 10(-7) M) had no effect on contractions evoked by transmural electrical stimulation (TES). However, both R396 (1 x 10(-5) to 1 x 10(-4) M) and MEN 10,376 (1 x 10(-6) to 1 x 10(-5) M) caused blockade of responses to TES. CP 96,345 (3 x 10(-7) M) antagonized TES-induced contractions only when studied after substantial blockade by R396 or MEN 10,376. Contractions to TES were not abolished by R396, MEN 10,376 or a combination of these antagonists with CP 96,345. R396 (1 x 10(-6) M) increased the maximum contraction to TES and potentiated the frequency-response curve in bronchi treated with MEN 10,376 (1 x 10(-6) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Electrical slow-wave activity from the circular layer of cat terminal antrum.

Intracellular recording techniques were used to characterize the electrical slow-wave activity through the thickness of the circular muscle layer of the cat terminal antrum. Muscle strips were pinned out in cross section to the floor of a recording chamber perfused with Krebs buffer. Circular muscle cells from the myenteric to the submucosal border then were impaled with 20- to 40-M omega glass microelectrodes, and slow-wave activity was recorded. Slow waves from the myenteric side of the circular layer consisted of an upstroke depolarization, a prominent plateau phase, and a downstroke repolarization. Slow-wave characteristics for cells along the myenteric border were Em, -74.2 +/- 1.3 mV; duration, 5.3 +/- 0.5 s; upstroke amplitude, 29.4 +/- 3.4 mV; upstroke velocity, 0.20 +/- 0.03 V/s; and frequency, 5.8 +/- 0.5/min. Slow waves from muscle cells along the submucosal side of the preparation lacked a discernible plateau phase. Slow waves from submucosal border cells had the following characteristics: Em, -80.4 +/- 1.4 mV (P less than 0.01); duration, 3.5 +/- 0.4 s (P less than 0.01); upstroke amplitude, 44.0 +/- 2.4 mV (P less than 0.01); upstroke velocity, 0.56 +/- 0.06 V/s (P less than 0.01); and frequency, 4.2 +/- 0.4/min (P less than 0.05). Slow waves were not affected by 10(-7)M tetrodotoxin and 10(-6)M atropine or by removal of the longitudinal muscle layer. Slow-wave activity within each region was maintained after dissecting the circular layer into submucosal and myenteric segments. The results suggest that two distinct slow waves exist within the circular muscle layer of the cat terminal antrum.(ABSTRACT TRUNCATED AT 250 WORDS)

Contribution of intracellular calcium to gallbladder smooth muscle contraction.

Studies were performed to evaluate the contribution of intracellular Ca2+ to gallbladder smooth muscle contraction under acetylcholine (ACh) or potassium stimulation. Gallbladder smooth muscle strips from adult guinea pigs were placed in tissue baths containing N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered physiological salt solution (PSS) and set to optimal length for contraction (Lo). The results were as follows, 1) A 20-min equilibration in zero Ca2(+)-0.1 mM ethylene glycol-bis( beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) PSS virtually abolished the response to potassium but not to ACh. 2) Substitution of strontium, an inhibitor of intracellular Ca2+ release, for Ca2+ significantly decreased the contractile response to ACh (3 X 10(-5), 10(-4), and 3 X 10(-4) M). Strontium had no effect on the response to 40 and 80 mM potassium. 3) Intracellular Ca2+ depletion significantly decreased gallbladder smooth muscle contraction to ACh (10(-4) M) but had no effect on the response to potassium (80 mM). 4) Ryanodine, a compound that inhibits Ca2+ storage by the sarcoplasmic reticulum, significantly decreased the contractile response to ACh (10(-4) M) but not to potassium (80 mM). These data support the observation that the use of intracellular Ca2+ by gallbladder smooth muscle for contraction is agonist dependent.