The selective phosphodiesterase 4 inhibitor roflumilast and phosphodiesterase 3/4 inhibitor pumafentrine reduce clinical score and TNF expression in experimental colitis in mice.

The specific inhibition of phosphodiesterase (PDE)4 and dual inhibition of PDE3 and PDE4 has been shown to decrease inflammation by suppression of pro-inflammatory cytokine synthesis. We examined the effect of roflumilast, a selective PDE4 inhibitor marketed for severe COPD, and the investigational compound pumafentrine, a dual PDE3/PDE4 inhibitor, in the preventive dextran sodium sulfate (DSS)-induced colitis model. The clinical score, colon length, histologic score and colon cytokine production from mice with DSS-induced colitis (3.5% DSS in drinking water for 11 days) receiving either roflumilast (1 or 5 mg/kg body weight/d p.o.) or pumafentrine (1.5 or 5 mg/kg/d p.o.) were determined and compared to vehicle treated control mice. In the pumafentrine-treated animals, splenocytes were analyzed for interferon-γ (IFNγ) production and CD69 expression. Roflumilast treatment resulted in dose-dependent improvements of clinical score (weight loss, stool consistency and bleeding), colon length, and local tumor necrosis factor-α (TNFα) production in the colonic tissue. These findings, however, were not associated with an improvement of the histologic score. Administration of pumafentrine at 5 mg/kg/d alleviated the clinical score, the colon length shortening, and local TNFα production. In vitro stimulated splenocytes after in vivo treatment with pumafentrine showed a significantly lower state of activation and production of IFNγ compared to no treatment in vivo. These series of experiments document the ameliorating effect of roflumilast and pumafentrine on the clinical score and TNF expression of experimental colitis in mice

In Vitro and in Vivo Anti-Inflammatory Activity of the New Glucocorticoid Ciclesonide

ABSTRACT The glucocorticoid ciclesonide is the 2ЈR-epimer of 2Ј-cyclohexyl-11␤-hydroxy-21-isobutyryloxy-16bH-dioxolo [5Ј,4Ј:16,17]pregna-1,4-diene-3,20-dione. The active metabolite desisobutyrylciclesonide (des-CIC) is derived from ciclesonide by esterase cleavage of isobutyrate at the C21 position. The relative binding affinities at the rat glucocorticoid receptor were dexamethasone, 100; ciclesonide, 12; des-CIC, 1212; and budesonide, 905. Des-CIC potently inhibited the activation of murine and human lymphocytes in a series of different in vitro systems. With the exception of concanavalin A-stimulated rat spleen cells, des-CIC was more potent than the parent compound. Des-CIC compared well with budesonide in all in vitro systems. Furthermore, the respective 2ЈS-epimers were always significantly less potent than the 2ЈR-epimers. In vivo, ciclesonide (intratracheal administration), des-CIC, and budesonide inhibited antigen-induced accumulation of eosinophils, protein, and tumor necrosis factor-␣ into the bronchoalveolar lavage fluid of ovalbumin-sensitized and -challenged Brown Norway rats with an ED 50 value ranging from 0.4 to 1.3 mg/kg, indicating similar potency, which suggests in vivo activation of the parent compound. Ciclesonide and budesonide inhibited the bradykinin-induced protein leakage into the rat trachea. In the rat cotton pellet model, ciclesonide inhibited granuloma formation (ED 50 :ϭ of 2 g/pellet), whereas budesonide and des-CIC were 15-and 20-fold less active; thymus involution was induced with an ED 50 of 303, 279, and 154 g/pellet, respectively. When applied orally to rats for 28 days, ciclesonide showed low potency in reducing weight of thymus and adrenals, suggesting low oral bioavailability. The in vivo data on ciclesonide highlight its effective local action and a reduced potential for side effects

Roflumilast in moderate-to-severe chronic obstructive pulmonary disease treated with longacting bronchodilators: two randomised clinical trials

Background Patients with chronic obstructive pulmonary disease (COPD) have few options for treatment. The efficacy and safety of the phosphodiesterase-4 inhibitor roflumilast have been investigated in studies of patients with moderate-to-severe COPD, but not in those concomitantly treated with longacting inhaled bronchodilators. The effect of roflumilast on lung function in patients with COPD that is moderate to severe who are already being treated with salmeterol or tiotropium was investigated. Methods In two double-blind, multicentre studies done in an outpatient setting, after a 4-week run-in, patients older than 40 years with moderate-to-severe COPD were randomly assigned to oral roflumilast 500 mu g or placebo once a day for 24 weeks, in addition to salmeterol (M2-127 study) or tiotropium (M2-128 study). The primary endpoint was change in prebronchodilator forced expiratory volume in 1s (FEV(1)). Analysis was by intention to treat. The studies are registered with ClinicalTrials.gov, number NCT00313209 for M2-127, and NCT00424268 for M2-128. Findings In the salmeterol plus roflumilast trial, 466 patients were assigned to and treated with roflumilast and 467 with placebo; in the tiotropium plus roflumilast trial, 371 patients were assigned to and treated with roflumilast and 372 with placebo. Compared with placebo, roflumilast consistently improved mean prebronchodilator FEV(1) by 49 mL (p<0.0001) in patients treated with salmeterol, and 80 mL (p<0.0001) in those treated with tiotropium. Similar improvement in postbronchodilator FEV(1) was noted in both groups. Furthermore, roflumilast had beneficial effects on other lung function measurements and on selected patient-reported outcomes in both groups. Nausea, diarrhoea, weight loss, and, to a lesser extent, headache were more frequent in patients in the roflumilast groups. These adverse events were associated with increased patient withdrawal. Interpretation Roflumilast improves lung function in patients with COPD treated with salmeterol or tiotropium, and could become an important treatment for these patients

Isolation and characterization of rat primary lung cells

Lung cell culture may be useful as anin vitro alternative to study the susceptibility of the lung to various toxic agents. Lungs from female Wistar rats were enzymatically digested by recirculating perfusion through the pulmonary artery with a sequence of solutions containing deoxyribonuclease, chymopapain, pronase, collagenase, and elastase. Lung tissue was microdissected and resuspended and the cells obtained were washed by centrifugation. By this isolation method, 2×108 cells per rat lung were obtained with an average viability of 97%. Lung cells cultured in medium containing antibiotics and serum maintained a viability of >70% for 5 d. Rat primary lung cells were exposed to various toxic agents and their viability was assessed by formazan production capacity after 18 h of incubation. Compared to rat and mouse hepatocyte cultures (EC50=5.8 mM), rat primary lung cells were much more susceptible to hydrogen peroxide (EC50=0.6 mM). All cell types were equally sensitive to the more potent toxicanttert-butylhydroperoxide (EC50=0.1 mM). Paraquat was more toxic to lung cells (EC50=0.03 mM) than to rat (EC50=2.8 mM) and mouse (EC50=0.2 mM) hepatocytes. In contrast, rat lung cells were less sensitive to sodium nitroprusside (EC50=2.6 mM) compared to rat (EC50=0.2 mM) and mouse (EC50=0.03 mM) hepatocytes. Nitrofurantoin and menadione (at EC50=0.04 mM and 0.006 mM, respectively) were more toxic to rat lung and liver cells than to murine hepatocytes (EC50=0.2 mM and 0.04 mM, respectively). Our findings demonstrate the applicability of this rat primary lung cell culture for studying the effects of lung toxicants

Reduction of colonic mucosa TNFα content by roflumilast.

<p>Mice were exposed to 3.5% DSS in drinking water for eleven days and were treated with roflumilast (either 1 or 5 mg/kg/d orally once daily for 11 days, n = 8) or 4% methocel (n = 5). At day 11 the colon was removed, weighed, vortexed in PBS and centrifuged. TNFα was quantified in the eluate by ELISA. Values represent mean ± SEM; *p<0.05.</p

Effect of roflumilast on clinical score and colon length. A. Mitigation of DSS-induced colitis by roflumilast.

<p>Mice were exposed to 3.5% DSS in drinking water for 11 days. Either 1 mg/kg/d roflumilast, 5 mg/kg/d roflumilast or 4% methocel were administered orally once daily for 11 days (n = 8). Non-DSS-treated mice received 5 mg/kg/d roflumilast (n = 8) or 4% methocel (n = 5). The degree of colitis was quantified by the clinical score assessing weight loss, stool consistency and rectal bleeding (range from 0 =  healthy to 4 =  maximal disease activity). Scores are depicted as mean ± SEM; *p<0.05, **p<0.01 versus DSS+methocel. <b>B. Effect of roflumilast on colon length shortening in DSS-induced colitis.</b> Mice were exposed to 3.5% DSS in drinking water for an 11 day period. Roflumilast treatment (either 1 mg/kg/d or 5 mg/kg/d orally, once daily for eleven days) or 4% methocel were started on the same day as DSS administration (n = 8). Non-DSS mice received 5 mg/kg/d roflumilast (n = 8) or 4% methocel (n = 5). Values are depicted as mean ± SEM. **p<0.01, ***p<0.001 versus DSS+methocel.</p

Relative improvement of experimental colitis with roflumilast 5 mg/kg/d and pumafentrine 5 mg/kg/d.

<p>Data is depicted as mean ± standard error * p<0.05,</p>**<p>p<0.01,</p>***<p>p<0.001, DSS: Dextrane sodium sulfate; TNFα: tumor necrosis factor-α.</p><p>Data are depicted as % improvement of the dextrane sulfate sodium (DSS) treatment groups versus DSS methocel groups.</p