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

Rieder F, Siegmund B, Bundschuh DS, Lehr HA, Endres S, Eigler A - PLoS ONE (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Clinical Pharmacology and Section of Gastroenterology, University of Munich, Munich, Germany.

ABSTRACT

Objective: 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.

Methods: 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.

Results: 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.

Conclusions: These series of experiments document the ameliorating effect of roflumilast and pumafentrine on the clinical score and TNF expression of experimental colitis in mice.

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Systemic effects of pumafentrine. A Effect of in vivo administered pumafentrine on PMA/ionomycine-induced IFNγ synthesis by splenocytes from DSS-exposed mice.Mice were exposed to 3.5% DSS in drinking water for 11 days and were treated with pumafentrine 5 mg/kg/d (n = 8) orally once daily for 11 days) or 4% methocel (n = 8). Non-DSS mice received 20 mg/kg/d pumafentrine (n = 8). After day 11 spleens were removed aseptically and splenocytes were isolated. After a 20 h incubation period with PMA 25 ng/ml and ionomycine 500 ng/ml, the experiment was stopped by three freeze-thaw cycles and total IFNγ was measured by ELISA. Results are depicted as mean ± SEM. *p<0.05 versus DSS+methocel. B. Effect of in vivo administered pumafentrine on PMA/ionomycine-induced CD69 expression by splenocytes from DSS-exposed mice. Mice were exposed to 3.5% DSS in drinking water for 11 days and were treated with pumafentrine 5 mg/kg/d (n = 8) orally once daily for 11 days) or 4% methocel (n = 8). Non-DSS mice received 20 mg/kg/d pumafentrine (n = 8). After day 11 spleens were removed aseptically and splenocytes were isolated and stimulated with PMA 25 ng/ml and ionomycine 500 ng/ml. After 20 h incubation period the CD69-expression was determined by flow cytometry. Bars represent the mean ± SEM of CD69 positive splenocytes. *p<0.05 versus DSS+methocel.
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pone-0056867-g006: Systemic effects of pumafentrine. A Effect of in vivo administered pumafentrine on PMA/ionomycine-induced IFNγ synthesis by splenocytes from DSS-exposed mice.Mice were exposed to 3.5% DSS in drinking water for 11 days and were treated with pumafentrine 5 mg/kg/d (n = 8) orally once daily for 11 days) or 4% methocel (n = 8). Non-DSS mice received 20 mg/kg/d pumafentrine (n = 8). After day 11 spleens were removed aseptically and splenocytes were isolated. After a 20 h incubation period with PMA 25 ng/ml and ionomycine 500 ng/ml, the experiment was stopped by three freeze-thaw cycles and total IFNγ was measured by ELISA. Results are depicted as mean ± SEM. *p<0.05 versus DSS+methocel. B. Effect of in vivo administered pumafentrine on PMA/ionomycine-induced CD69 expression by splenocytes from DSS-exposed mice. Mice were exposed to 3.5% DSS in drinking water for 11 days and were treated with pumafentrine 5 mg/kg/d (n = 8) orally once daily for 11 days) or 4% methocel (n = 8). Non-DSS mice received 20 mg/kg/d pumafentrine (n = 8). After day 11 spleens were removed aseptically and splenocytes were isolated and stimulated with PMA 25 ng/ml and ionomycine 500 ng/ml. After 20 h incubation period the CD69-expression was determined by flow cytometry. Bars represent the mean ± SEM of CD69 positive splenocytes. *p<0.05 versus DSS+methocel.

Mentions: As dual selective PDE3/PDE4 inhibitors have never been tested in a preventive colitis model we evaluated the effects of in vivo-administered pumafentrine on IFN-γ production in vitro. Splenocytes at the end of day 11 were cultured for 20 h with PMA (25 ng/ml) plus ionomycine (500 ng/ml) or without stimulus. PMA plus ionomycine stimulated splenocytes from the methocel-treated DSS group showed a higher IFNγ production as compared to the pumafentrine-treated animals (Figure 6A). Controls not exposed to DSS showed the highest IFNγ secretion (20 mg/kg/d pumafentrine group). In preliminary experiments no significant difference in IFNγ production between control animals receiving pumafentrine or water was detected. The unstimulated splenocytes did not produce detectable levels of IFNγ (data not shown).


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

Rieder F, Siegmund B, Bundschuh DS, Lehr HA, Endres S, Eigler A - PLoS ONE (2013)

Systemic effects of pumafentrine. A Effect of in vivo administered pumafentrine on PMA/ionomycine-induced IFNγ synthesis by splenocytes from DSS-exposed mice.Mice were exposed to 3.5% DSS in drinking water for 11 days and were treated with pumafentrine 5 mg/kg/d (n = 8) orally once daily for 11 days) or 4% methocel (n = 8). Non-DSS mice received 20 mg/kg/d pumafentrine (n = 8). After day 11 spleens were removed aseptically and splenocytes were isolated. After a 20 h incubation period with PMA 25 ng/ml and ionomycine 500 ng/ml, the experiment was stopped by three freeze-thaw cycles and total IFNγ was measured by ELISA. Results are depicted as mean ± SEM. *p<0.05 versus DSS+methocel. B. Effect of in vivo administered pumafentrine on PMA/ionomycine-induced CD69 expression by splenocytes from DSS-exposed mice. Mice were exposed to 3.5% DSS in drinking water for 11 days and were treated with pumafentrine 5 mg/kg/d (n = 8) orally once daily for 11 days) or 4% methocel (n = 8). Non-DSS mice received 20 mg/kg/d pumafentrine (n = 8). After day 11 spleens were removed aseptically and splenocytes were isolated and stimulated with PMA 25 ng/ml and ionomycine 500 ng/ml. After 20 h incubation period the CD69-expression was determined by flow cytometry. Bars represent the mean ± SEM of CD69 positive splenocytes. *p<0.05 versus DSS+methocel.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585290&req=5

pone-0056867-g006: Systemic effects of pumafentrine. A Effect of in vivo administered pumafentrine on PMA/ionomycine-induced IFNγ synthesis by splenocytes from DSS-exposed mice.Mice were exposed to 3.5% DSS in drinking water for 11 days and were treated with pumafentrine 5 mg/kg/d (n = 8) orally once daily for 11 days) or 4% methocel (n = 8). Non-DSS mice received 20 mg/kg/d pumafentrine (n = 8). After day 11 spleens were removed aseptically and splenocytes were isolated. After a 20 h incubation period with PMA 25 ng/ml and ionomycine 500 ng/ml, the experiment was stopped by three freeze-thaw cycles and total IFNγ was measured by ELISA. Results are depicted as mean ± SEM. *p<0.05 versus DSS+methocel. B. Effect of in vivo administered pumafentrine on PMA/ionomycine-induced CD69 expression by splenocytes from DSS-exposed mice. Mice were exposed to 3.5% DSS in drinking water for 11 days and were treated with pumafentrine 5 mg/kg/d (n = 8) orally once daily for 11 days) or 4% methocel (n = 8). Non-DSS mice received 20 mg/kg/d pumafentrine (n = 8). After day 11 spleens were removed aseptically and splenocytes were isolated and stimulated with PMA 25 ng/ml and ionomycine 500 ng/ml. After 20 h incubation period the CD69-expression was determined by flow cytometry. Bars represent the mean ± SEM of CD69 positive splenocytes. *p<0.05 versus DSS+methocel.
Mentions: As dual selective PDE3/PDE4 inhibitors have never been tested in a preventive colitis model we evaluated the effects of in vivo-administered pumafentrine on IFN-γ production in vitro. Splenocytes at the end of day 11 were cultured for 20 h with PMA (25 ng/ml) plus ionomycine (500 ng/ml) or without stimulus. PMA plus ionomycine stimulated splenocytes from the methocel-treated DSS group showed a higher IFNγ production as compared to the pumafentrine-treated animals (Figure 6A). Controls not exposed to DSS showed the highest IFNγ secretion (20 mg/kg/d pumafentrine group). In preliminary experiments no significant difference in IFNγ production between control animals receiving pumafentrine or water was detected. The unstimulated splenocytes did not produce detectable levels of IFNγ (data not shown).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Clinical Pharmacology and Section of Gastroenterology, University of Munich, Munich, Germany.

ABSTRACT

Objective: 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.

Methods: 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.

Results: 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.

Conclusions: These series of experiments document the ameliorating effect of roflumilast and pumafentrine on the clinical score and TNF expression of experimental colitis in mice.

Show MeSH
Related in: MedlinePlus