LPDE4 (IC50 = 100 nM); HPDE4 (IC50 = 120 nM)

Human neutrophil functions are inhibited by cipolast (0.1 nM–10 μM; 5 min)[2]. A 5-minute exposure to cipomalost (0.1 nM–10 μM) inhibits the eosinophil chemiluminescence response[2]. Cilomilast (0.001-100 μM; 30 min) prevents human monocytes and human whole blood from synthesizing TNFα[2].

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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. [2]

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First-generation phosphodiesterase 4 (PDE4) inhibitors, such as rolipram, inhibit the activation of immune and inflammatory cells. The clinical use of these compounds is limited by gastrointestinal side effects, such as increased acid secretion and nausea. Consequently, the challenge has been to design novel PDE4 inhibitors that maintain the anti-inflammatory actions of rolipram while achieving an improved side effect profile. Among the first of this new class of PDE4 inhibitors specifically designed to have an improved therapeutic index relative to earlier compounds is Cilomilast/SB 207499 (Ariflo) [c-4-cyano-4-(3-cyclopentyloxy-4-methoxy-phenyl)-r-1-cyclohexanecarboxyl ic acid]. In this study, we compared the anti-inflammatory and gastric secretogogue activities of Cilomilast/SB 207499 with those of rolipram. The cellular models used were (1) histamine release from human basophils, (2) tumor necrosis factor-alpha generation in human monocytes, (3) degranulation of human neutrophils, (4) antigen-driven proliferation and cytokine synthesis from human T cells and (5) acid secretion from isolated rabbit gastric glands. SB 207499 inhibited the activation of a variety of immune and inflammatory cells in a concentration-dependent manner: (1) histamine release in basophils [-log IC25 = 6.6 +/- 0.3 vs. 8.0 for (R)-rolipram], (2) lipopolysacchride-induced TNF-alpha formation in monocytes [-log IC50 = 7.0 +/- 0.1 vs. 7.2 +/- 0.1 for (R)-rolipram], (3) fMLP-induced degranulation in neutrophils [-log IC15 = 7.1 +/- 0.2 vs. 6.4 +/- 0.5 for (R)-rolipram], (4) house dust mite induced-proliferation of peripheral blood mononuclear cells [-log IC40 = 6.5 +/- 0.3 vs. 6.4 +/- 0.3 for (R)-rolipram] and (5) ragweed-induced production of interferon-gamma [-log IC50 = 5.4] and interleukin-5 [-log IC50 = 5.0]. Although SB 207499 inhibits the activation of a variety of immune and inflammatory cells with a potency equal to that of rolipram, it is > 100-fold less potent than the latter compound as an acid secretagogue [-log EC50 = 6.1 +/- 0.1 vs. 8.3 +/- 0.2 for (R)-rolipram]. Collectively, these data indicate that SB 207499 retains the anti-inflammatory activity of the prototypical PDE4 inhibitor rolipram but is substantially less likely to stimulate gastric acid secretion. [3]

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A rapid and efficient method to stimulate bone regeneration would be useful in orthopaedic stem cell therapies. Rolipram is an inhibitor of phosphodiesterase 4 (PDE4), which mediates cyclic adenosine monophosphate (cAMP) degradation. Systemic injection of rolipram enhances osteogenesis induced by bone morphogenetic protein 2 (BMP-2) in mice. However, there is little data on the precise mechanism, by which the PDE4 inhibitor regulates osteoblast gene expression. In this study, we investigated the combined ability of BMP-2 and Cilomilast, a second-generation PDE4 inhibitor, to enhance the osteoblastic differentiation of mesenchymal stem cells (MSCs). The alkaline phosphatase (ALP) activity of MSCs treated with PDE4 inhibitor (Cilomilast or rolipram), BMP-2, and/or H89 was compared with the ALP activity of MSCs differentiated only by osteogenic medium (OM). Moreover, expression of Runx2, osterix, and osteocalcin was quantified using real-time polymerase chain reaction (RT-PCR). It was found that cilomilast enhances the osteoblastic differentiation of MSCs equally well as rolipram in primary cultured MSCs. Moreover, according to the H89 inhibition experiments, Smad pathway was found to be an important signal transduction pathway in mediating the osteogenic effect of BMP-2, and this effect is intensified by an increase in cAMP levels induced by PDE4 inhibitor. [4]

In mice, SB-207499 (1-100 mg/kg; po) substantially and dose-dependently suppresses human TNFα production[1]. With an ED50 of 2.3 mg/kg, SB-207499 (0.1-100 mg/kg; oral gavage) restores reserpine-induced hypothermia in mice[1]. In mice, intralesional IL-4 concentrations and the chronic oxazolone-induced inflammatory response are inhibited by SB-207499 (500 μg/ear; bid for 6 d)[1].

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The ability of the second generation phosphodiesterase 4 inhibitor Cilomilast/SB 207499 (Ariflo), [c-4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)-r-l-cyclohexane carboxylic acid], to inhibit inflammatory cytokine production in vivo was evaluated and compared to that of rolipram, a first generation phosphodiesterase 4 inhibitor. To examine human tumor necrosis factor alpha (TNFalpha) production, human monocytes were adoptively transferred into Balb/c mice and challenged with lipopolysaccharide (LPS). In this model, SB 207499 inhibited human TNFalpha production with oral ED50 of 4.9 mg/kg. Similarly, R-rolipram inhibited human TNFalpha production with an ED50 of 5.1 mg/kg, p.o. In contrast to their equipotent activity against TNFalpha production, SB 207499 (ED50 = 2.3 mg/kg, p.o.) was 10-fold less potent than R-rolipram (ED50 = 0.23 mg/kg, p.o.) in reversing reserpine-induced hypothermia, a model of antidepressant activity. In time course studies, SB 207499 (30 mg/kg, p.o.) inhibited TNFalpha production for at least 10 hr; substantial plasma concentrations of SB 207499 were detected over the same interval. The ability of Cilomilast/SB 207499 to modulate interleukin-4 production in vivo was assessed in a chronic oxazolone-induced contact sensitivity model in Balb/c mice. In this model, topical administration of SB 207499 (1000 microgram) inhibited intralesional concentrations of interleukin-4 (55%; P <.01). The results demonstrate that SB 207499 is a potent inhibitor of inflammatory cytokine production in a variety of settings in vivo. Moreover, although it is as potent as R-rolipram in inhibiting TNFalpha production, it has substantially less central nervous system activity. Thus SB 207499 represents an excellent candidate with which to evaluate the antiinflammatory potential of PDE4 inhibitors. [1]

Preparation of bone marrow cells and cell cultures [4]
Rat stromal cells were isolated according to the protocol of Kopen et al. Bone marrow was obtained from the tibias and femurs of male Wistar rats (4 weeks old) by flushing femurs and tibias with α-MEM medium and penicillin/streptomycin (50 U/mL and 50 mg/mL, respectively) supplemented with heparin. Cells were then washed in medium without heparin, centrifuged, and plated in a dish coated with fibronectin. One day later, non-adherent cells were removed by 3 washes with PBS. Adherent cells were further cultured in standard medium for 3 days. MSCs were grown in SM at 37 °C in a 5% CO2 atmosphere. The medium was change twice a week, and a subculture was performed every week. At passage 3, the cells were trypsinised and plated at a density of 38,000/cm2 for use in experiments. After 3 days, medium, with and without reagents, was added. The treatment was performed for 11 days (or 21 days for Alizarin Red staining), changing the medium every 3 days.

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RNA preparation and RT-PCR [4]
Total RNA was isolated from cultured cells in the presence of PDE4 inhibitors, BMP-2, standard and osteogenic medium by using QuickGene-Mini80, according to the manufacturer’s instructions. Total RNA (1 μg) was reverse-transcribed using the SuperScript™ III CellsDirect cDNA Synthesis system. The reaction program was 10 min at 25 °C, 2 h at 37 °C, and 5 s at 85 °C. Aliquots of the cDNA were subjected to PCR and amplified in a 20-μL reaction mixture using SYBR® Green Real-time PCR Master Mix. Amplifications were performed in StepOne Plus with an initial denaturation step at 95 °C for 10 min, followed by 35 cycles of denaturation at 95 °C for 15 s, annealing for 60 s at the specified temperature, and extension at 72 °C for 60 s. The PCR primer sequences and annealing temperatures are presented in Table 1.

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Assay for alkaline phosphatase activity [4]
Cell cultures were processed for ALP activity after 11 days of treatment with the reagents. Intracellular ALP activity was measured using the TRACP & ALP Assay Kit. Cells were washed 3 times with PBS, and then, lysed with 500 μL of lysis buffer containing physiological saline and 1% NP-40. Lysates were also prepared from cells grown in DMSO, standard and osteogenic medium as controls. The cells lysates were then mixed with an assay mixture containing p-nitrophenyl phosphate and incubated at 37 °C for 30 min, at which time the reaction was stopped by the addition of 0.4 M NaOH. After incubation, the amount of p-nitrophenol released by the reaction was measured with a spectrophotometer at 405 nm. All values were normalised against cell number. Protein in the cell lysates was determined using the micro Bio-Rad Protein Assay kit. Data were expressed as a ratio of ALP activity per milligram of protein.

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Alizarin Red staining Cells were washed twice with PBS and fixed in 10% formalin for 10 min. The cells were then stained with 1% Alizarin Red solution for 10 min at 37 °C and washed 3 times with PBS.

Animal/Disease Models: Male balb/c (Bagg ALBino) mouse (18-25 g) are injected with human monocytes and LPS[1]
Doses: 1, 5, 10, 50, 100 mg/kg
Route of Administration: Po after the injection of human monocytes and before LPS challenge
Experimental Results: Inhibited the production of human TNFα, with an ED50 of 4.9 mg/kg.

[1]. SB 207499 (Ariflo), a second generation phosphodiesterase 4 inhibitor, reduces tumor necrosis factor alpha and interleukin-4 production in vivo. J Pharmacol Exp Ther. 1998 Nov;287(2):705-11.

[2]. Anti-inflammatory and immunomodulatory potential of the novel PDE4 inhibitor roflumilast in vitro. J Pharmacol Exp Ther. 2001 Apr;297(1):267-79.

[3]. SB 207499 (Ariflo), a potent and selective second-generation phosphodiesterase 4 inhibitor: in vitro anti-inflammatory actions. J Pharmacol Exp Ther. 1998 Jan;284(1):420-6.

[4]. Cilomilast enhances osteoblast differentiation of mesenchymal stem cells and bone formation induced by bone morphogenetic protein 2. Biochimie. 2012 Nov;94(11):2360-5.

4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)-1-cyclohexanecarboxylic acid is a member of methoxybenzenes.
Cilomilast (Ariflo, SB-207,499) is a drug which was developed for the treatment of respiratory disorders such as asthma and Chronic Obstructive Pulmonary Disease (COPD). It is orally active and acts as a selective Phosphodiesterase-4 inhibitor. Following four clinical trials, the drug proved to be effective in treating COPD, however it has never been marketed due to a poor side effect profile.

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Drug Indication
Investigated for use/treatment in chronic obstructive pulmonary disease (COPD).

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Mechanism of Action
Cilomilast shows high selectivity for cAMP-specific PDE4, an isoenzyme that predominates in pro-inflammatory and immune cells and that is 10-fold more selective for PDE4D than for PDE4A, -B or -C. In vitro, Cilomilast suppresses the activity of several pro-inflammatory and immune cells that have been implicated in the pathogenesis of asthma and COPD. Moreover, it is highly active in animal models of these diseases. Cilomilast has been shown to exert potent anti-inflammatory effects both in vitro and in vivo.

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In this study, we investigated osteoblast differentiation in the combined presence of BMP-2 and PDE4 inhibitors. Our results support the notions that signal transduction via the cAMP pathway is necessary but not sufficient to induce MSC differentiation. In fact, PDE4 inhibitor alone did not induce a complete differentiation of MSCs, even though the PDE4 inhibitor could enhance the effect of BMP-2. Moreover, we demonstrated that the cAMP pathway is not involved in the differentiation of primary MSC induced by BMP-2 alone but is critical in the enhancement of bone formation in the presence of PDE4 inhibitors and BMP-2. We have shown that Cilomilast, a second-generation PDE4 inhibitor, could be a useful new tool in orthopaedic stem cell therapies, because it is able to induce rapid mineralisation in the presence of BMP-2. [4]

C20H25NO4

343.42

343.178

C, 69.95; H, 7.34; N, 4.08; O, 18.64

153259-65-5

151170

White to off-white solid powder

1.2±0.1 g/cm3

549.1±50.0 °C at 760 mmHg

157ºC

285.9±30.1 °C

0.0±1.6 mmHg at 25°C

1.568

3.3

1

5

5

25

511

0

COC1=C(C=C(C=C1)C2(CCC(CC2)C(=O)O)C#N)OC3CCCC3

CFBUZOUXXHZCFB-LDTOLXSISA-N

InChI=1S/C20H25NO4/c1-24-17-7-6-15(12-18(17)25-16-4-2-3-5-16)20(13-21)10-8-14(9-11-20)19(22)23/h6-7,12,14,16H,2-5,8-11H2,1H3,(H,22,23)/t14-,20+

cis-4-Cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexanecarboxylic acid

Cilomilast; SB 207499; Cilomilast; 153259-65-5; Ariflo; SB-207,499; SB 207,499; SB207,499; Cilomilast [USAN:INN]; Ariflo (TN); SB207499; Ariflo; SB-207499;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.28 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.28 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.28 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 5% DMSO+95% Corn oil:30 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)