Piclamilast

Selective inhibition of purified human phosphodiesterase 4A expressed in yeast cell GL62 by ciclamilast, piclamilast, and rolipram.[Pubmed:15339393]

Acta Pharmacol Sin. 2004 Sep;25(9):1171-5.

AIM: To improve the specific activity of human phosphodiesterase 4A (PDE4A) expressed in yeast cell GL62 and investigate the effects of selective phosphodiesterase 4 (PDE4) inhibitors (ciclamilast, Piclamilast, and rolipram), selective phosphodiesterase 5 (PDE5) inhibitor zaprinast, and cyclooxygenase (COX) inhibitors (aspirin, indomethacin) on human PDE4A activity expressed in yeast cell GL62. METHODS: Human PDE4A was expressed in yeast cell GL62 after CuSO4 induction and the specific activity of human PDE4A was improved by ammonium sulfate fractionation, DEAE Sephadex A-50 chromatography, and Sephadex G-100 chromatography. The activity of PDE4A was measured by high performance liquid chromatography (HPLC). RESULTS: Induced PDE4A activity expressed in crude yeast cell GL62 supernatant and pellet was (340+/-21) nmol/g/min and (250+/-25) nmol/g/min respectively. The specific activity of recombinant PDE4A in supernatant was improved 6.4 fold. Ciclamilast, Piclamilast, and rolipram could inhibit PDE4A activity. The IC50 values (95 % confidence limits) of ciclamilast, Piclamilast, and rolipram were 1.27 (0.84-1.91), 66.4 (33.3-132.2), and 3.73 (2.51-5.53) micromol/L respectively. Zaprinast, aspirin, and indomethacin had no obvious inhibitory effect on PDE4A activity. CONCLUSION: The specific activity of PDE4A expressed in yeast cell GL62 can be improved by ammonium sulfate fractionation, DEAE Sephadex A-50 chromatography, and Sephadex G-100 chromatography. Ciclamilast, Piclamilast, and rolipram can inhibit PDE4A activity while zaprinast, aspirin, and indomethacin have no obvious inhibitory effect on PDE4A activity. Human PDE4A expressed in GL62 might be useful in the research and screening of new selective PDE4 inhibitors.

Phosphodiesterase IV inhibition by piclamilast potentiates the cytodifferentiating action of retinoids in myeloid leukemia cells. Cross-talk between the cAMP and the retinoic acid signaling pathways.[Pubmed:15292163]

J Biol Chem. 2004 Oct 1;279(40):42026-40.

Inhibition of phosphodiesterase IV by N-(3,5-dichloropyrid-4-yl)-3-cyclopentyloxy-4-methoxybenzamide (Piclamilast) enhances the myeloid differentiation induced by all-trans-retinoic acid (ATRA), retinoic acid receptor alpha (RARalpha), or retinoic acid receptor X agonists in NB4 and other retinoid-sensitive myeloid leukemia cell types. ATRA-resistant NB4.R2 cells are also partially responsive to the action of Piclamilast and retinoic acid receptor X agonists. Treatment of NB4 cells with Piclamilast or ATRA results in activation of the cAMP signaling pathway and nuclear translocation of cAMP-dependent protein kinase. This causes a transitory increase in cAMP-responsive element-binding protein phosphorylation, which is followed by down-modulation of the system. ATRA + Piclamilast have no additive effects on the modulation of the cAMP pathway, and the combination has complex effects on cAMP-regulated genes. Piclamilast potentiates the ligand-dependent transactivation and degradation of RARalpha through a cAMP-dependent protein kinase-dependent phosphorylation. Enhanced transactivation is also observed in the case of PML-RARalpha. In NB4 cells, increased transactivation is likely to be at the basis of enhanced myeloid maturation and enhanced expression of many retinoid-dependent genes. Piclamilast and/or ATRA exert major effects on the expression of cEBP and STAT1, two types of transcription factors involved in myeloid maturation. Induction and activation of STAT1 correlates directly with enhanced cytodifferentiation. Finally, ERK and the cAMP target protein, Epac, do not participate in the maturation program activated by ATRA + Piclamilast. Initial in vivo studies conducted in severe combined immunodeficiency mice transplanted with NB4 leukemia cells indicate that the enhancing effect of Piclamilast on ATRA-induced myeloid maturation translates into a therapeutic benefit.

Piclamilast inhibits the pro-apoptotic and anti-proliferative responses of A549 cells exposed to H(2)O(2) via mechanisms involving AP-1 activation.[Pubmed:22360706]

Free Radic Res. 2012 May;46(5):690-9.

AIMS: Reactive oxygen species (ROS) are involved in the pathogenesis of many inflammatory diseases such as chronic obstructive pulmonary disease (COPD). They can alter the expression of genes involved in cellular damage by activating transcription factors, including the NF-kappaB and the activator protein 1 (AP-1). Phosphodiesterase type 4 (PDE4) inhibitors have anti-inflammatory and antioxidant effects, as described in in vivo and in vitro COPD models. This study analysed the effects of Piclamilast, a selective PDE4 inhibitor, on modulating the global gene expression profile in A549 cells exposed to H(2)O(2). MAIN METHODS: Changes in gene expression were analysed using high-density Affymetrix microarrays and validated by RT-PCR. Cell proliferation was studied using BrdU incorporation. Apoptosis was assessed by flow cytometry using annexin V-fluorescein isothiocyanate. C-Jun phosphorylation and AP-1 activation were determined by ELISA and luciferase assay, respectively. KEY FINDINGS: Our results indicate that H(2)O(2) modified the expression of several genes related to apoptosis, cell cycle control and cell signalling, including IL8, FAS, HIG2, CXCL2, CDKN25 and JUNB. Piclamilast pre-treatment significantly inhibited the changes in 23 genes via mechanisms involving AP-1 activation and c-Jun phosphorylation at Ser63. Functional experiments confirmed our results, suggesting new targets related to the antioxidant properties of PDE4 inhibitors. SIGNIFICANCE: This is the first study to demonstrate antioxidant effects of a selective PDE4 inhibitor at the global gene expression level, and the results support the importance of AP-1 as a key regulator of the expression of genes involved in the inflammatory response of epithelial cells to oxidative damage.

Effects of piclamilast, a selective phosphodiesterase-4 inhibitor, on oxidative burst of sputum cells from mild asthmatics and stable COPD patients.[Pubmed:15765929]

Lung. 2004;182(6):369-77.

Oxidative stress associated with increased presence of neutrophils is an important feature of inflammatory airways diseases like asthma or chronic obstructive pulmonary disease. We studied the in vitro effect of Piclamilast (RP73401), a selective phosphodiesterase (PDE)-4 inhibitor, compared to theophylline and prednisolone, on respiratory burst of sputum cells from mild asthmatics and COPD patients. Sputum cells were harvested from mild asthmatics and stable COPD patients and treated with Piclamilast, theophylline or prednisolone. Respiratory burst was assessed by luminol-dependent chemoluminescence after stimulation with 10 microM n-formyl-met-leu-phe (FMLP). Piclamilast inhibited FMLP-induced respiratory burst of sputum cells in a concentration-dependent manner (asthma: EC50 approximately 100 nM, max. inhibition: 97.5+/-5% at 100 microM; COPD: EC50 approximately 1 microM, max. inhibition: 70.6+/-4.5% at 100 microM), whereas maximal inhibition observed with theophylline (asthma: max. inhib. 27+/-15%; COPD: 6+/-2%, both p < 0.05 vs. Piclamilast) and prednisolone (asthma: 16+/-6%; COPD: 7.8+/-6.2%, both p < 0.05 vs. Piclamilast) was weaker. Inhibition by Piclamilast was largely reversed through pretreatment of cells with the adenylcyclase inhibitor SQ22536. We concluded that Piclamilast, a selective PDE-4 inhibitor, attenuates the respiratory burst of sputum cells from mild asthmatics and COPD patients in vitro. These data underline the potential of PDE-4 inhibition as a novel therapeutic approach to inflammatory airway diseases like asthma or COPD.

Inhibitor binding to type 4 phosphodiesterase (PDE4) assessed using [3H]piclamilast and [3H]rolipram.[Pubmed:12704225]

J Pharmacol Exp Ther. 2003 May;305(2):565-72.

Piclamilast is a type 4 phosphodiesterase (PDE4) inhibitor with equal affinity for the high-affinity rolipram binding site (HARBS) and low-affinity rolipram binding site (LARBS). The binding of [(3)H]Piclamilast to preparations of rat brain and peripheral tissue was investigated and compared with that of [(3)H]rolipram. [(3)H]Piclamilast binding was high-affinity, saturable, reversible, and partially Mg(2+)-dependent. Binding was detected both to membrane and soluble fractions, with K(d) values of 3.1 and 4.5 nM, respectively. The B(max) values for [(3)H]Piclamilast were about 1.5-fold greater than that of [(3)H]rolipram binding, suggesting that [(3)H]Piclamilast, but not [(3)H]rolipram, binds to LARBS as well as the HARBS. The HARBS was present in all the brain regions examined, but not in peripheral tissues. All PDE4 inhibitors tested were potent competitors for [(3)H]Piclamilast binding; the competition curves for rolipram, desmethylPiclamilast, ICI 63,197, and Ro 20-1724 were better described by a two-site model, while the competition curves for Piclamilast, cilomilast, roflumilast, and CDP 840 were adequately described by a one-site model. Inhibitors of other PDE families were much less potent. The inhibition of [(3)H]Piclamilast was further tested in the presence of 1 microM rolipram to isolate the LARBS. Under this condition, the competition curves for all the inhibitors were adequately described by a one-site model, with K(i) values close to that for the LARBS. The results indicated that [(3)H]Piclamilast is a useful tool to directly study inhibitor interaction with the HARBS and the LARBS in rat brain.

Anti-inflammatory and immunomodulatory potential of the novel PDE4 inhibitor roflumilast in vitro.[Pubmed:11259554]

J Pharmacol Exp Ther. 2001 Apr;297(1):267-79.

From a series of benzamide derivatives, roflumilast (3-cyclo-propylmethoxy-4-difluoromethoxy-N-[3,5-di-chloropyrid-4-yl]-benzamide) was identified as a potent and selective PDE4 inhibitor. It inhibits PDE4 activity from human neutrophils with an IC(50) of 0.8 nM without affecting PDE1 (bovine brain), PDE2 (rat heart), and PDE3 and PDE5 (human platelets) even at 10,000-fold higher concentrations. Roflumilast is almost equipotent to its major metabolite formed in vivo (roflumilast N-oxide) and Piclamilast (RP 73401), however, more than 100-fold more potent than rolipram and Ariflo (cilomilast; SB 207499). The anti-inflammatory and immunomodulatory potential of roflumilast and the reference compounds was investigated in various human leukocytes using cell-specific responses: neutrophils [N-formyl-methyl-leucyl-phenylalanine (fMLP)-induced formation of LTB(4) and reactive oxygen species (ROS)], eosinophils (fMLP- and C5a-induced ROS formation), monocytes, monocyte-derived macrophages, and dendritic cells (lipopolysaccharide-induced tumor necrosis factor-alpha synthesis), and CD4+ T cells (anti-CD3/anti-CD28 monoclonal antibody-stimulated proliferation, IL-2, IL-4, IL-5, and interferon-gamma release). Independent of the cell type and the response investigated, the corresponding IC values (for half-maximum inhibition) of roflumilast were within a narrow range (2-21 nM), very similar to roflumilast N-oxide (3-40 nM) and Piclamilast (2-13 nM). In contrast, cilomilast (40-3000 nM) and rolipram (10-600 nM) showed greater differences with the highest potency for neutrophils. Compared with neutrophils and eosinophils, representing the terminal inflammatory effector cells, the relative potency of roflumilast and its N-oxide for monocytes, CD4+ T cells, and dendritic cells is substantially higher compared with cilomilast and rolipram, probably reflecting an improved immunomodulatory potential. The efficacy of roflumilast in vitro and in vivo (see accompanying article in this issue) suggests that roflumilast will be useful in the treatment of chronic inflammatory disorders such as asthma and chronic obstructive pulmonary disease.

Anti-inflammatory and bronchodilator properties of RP 73401, a novel and selective phosphodiesterase type IV inhibitor.[Pubmed:7889300]

Br J Pharmacol. 1994 Dec;113(4):1423-31.

1. We have investigated the effects of RP 73401, a novel, potent and highly selective cyclic nucleotide phosphodiesterase (PDE) type IV inhibitor, in guinea-pig and rat models of bronchoconstriction and allergic inflammation. In some models, the effects of RP 73401 have been compared with those of the standard PDE type IV inhibitor, rolipram. 2. RP 73401 (0.4-400 micrograms kg-1, intratracheally (i.t.) on lactose) inhibited antigen-induced bronchospasm in previously sensitized conscious guinea-pigs (ID50: 7 +/- 1 micrograms kg-1) and in anaesthetized rats (ID50: 100 +/- 25 micrograms kg-1). Rolipram inhibited the antigen-induced bronchospasm in guinea-pigs with an ID50 of 5 +/- 1 micrograms kg-1. In guinea-pig bronchoalveolar lavage (BAL) fluid, total inflammatory cell and eosinophil numbers were reduced by RP 73401 (ID50s: 3.9 +/- 0.8 micrograms kg-1 and 3.2 +/- 0.7 micrograms kg-1, respectively). In the rat, inflammatory cell numbers are less affected. Only the highest dose of RP 73401 (400 micrograms kg-1) significantly inhibited eosinophil influx (41 +/- 16% inhibition). 3. RP 73401 (0.02-100 micrograms kg-1, i.v.) inhibited PAF-induced bronchial hyperreactivity to bombesin in the anaesthetized guinea-pig (ID50: 0.09 +/- 0.03 micrograms kg-1) and inhibited (0.4-40 micrograms kg-1, i.t.) histamine-induced airway microvascular leakage in the anaesthetized guinea-pig by approximately 60% at all doses. 4. RP 73401 relaxed guinea-pig isolated trachea under basal tone (EC50: 9 nM) and when precontracted with histamine (IC50: 2 nM), methacholine (IC50: 29 nM) or leukotriene D4 (LTD4, IC50: 4 nM). 5. RP 73401 (0.4-100 microg kg-1, i.t.) inhibited bronchospasm induced by histamine (ID.%: 34 +/- 6 microg kg-1), methacholine (ID50: 66 +/- 12 pg kg-1) and LTD4 (ID50: <4 microg kg-1) in the anaesthetized guinea pig.Against these same bronchoconstrictors, rolipram (i.t.) had ID5o values of 44 +/- 4, 72 +/- 18 and<4 pg kg- respectively. RP 73401 (4 and 40 pg kg-, i.t.) increased the magnitude and duration of bronchodilatation produced by salbutamol in the anaesthetized guinea-pig. At doses producing significant bronchodilatation, RP 73401 was without effect on heart rate or blood pressure in the anaesthetized guinea-pig. RP 73401 (0.01 -0.25 mg kg-1, i.v.) did not affect heart rate and produced only a small fall in blood pressure in the anaesthetized rat.6. These data demonstrate that RP 73401 and rolipram inhibit antigen- and mediator-induced bronchospasmin guinea-pigs with the same potency. Furthermore, RP 73401 administered directly into the airways, protects against allergic airway inflammation. These results indicate the importance of PDE IV in regulating smooth muscle and inflammatory cell activity. At doses suppressing the inflammatory response in the lung, RP 73401 had little effect in the cardiovascular system. RP 73401 may have a role as a bronchodilator and, more importantly, as a prophylactic anti-inflammatory agent in the treatment of asthma.