PDE4 (IC50 = 0.2~0.9 nM)

Roflumilast is a subnanomolar inhibitor of the majority of PDE4 splice variants tested and has no effect on PDE enzymes other than PDE4. With the exception of PDE4C (4C1, IC50=3 nM; 4C2, IC50= 4.3 nM), which is inhibited with somewhat lesser potency, it does not demonstrate PDE4 isoform selectivity [2]. A strong and specific PDE4 inhibitor is roflumast. At concentrations up to 10,000-fold, roflumilast does not affect other PDE isoenzymes, such as PDE1, PDE2, PDE3, or PDE5. This makes it a monoselective inhibitor of PDE4. Roflumilast inhibits the activity of human neutrophils. Roflumilast prevents monocyte-derived dendritic cells from synthesizing TNFα. Cytokine synthesis and CD4+ T cell proliferation are inhibited by rolfumilast. Up to 60% of proliferation can be inhibited by rolumilast at a potency (IC30) of 7 nM [3].

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Roflumilast was identified in 1993 from a series of benzamides in a comprehensive screening programme. The high potency and selectivity of roflumilast for competitive inhibition of PDE4, without affecting PDE1, 2, 3 or 5 isoenzymes, from various cells and tissues has been reported previously. These early studies have been extended to a series of human recombinant PDE enzymes tested against PDE1–11 (Table 1). Results confirm that roflumilast does not affect any other PDE enzyme and is a subnanomolar inhibitor of most of the PDE4 splicing variants tested. Roflumilast shows no PDE4 subtype selectivity, with the exception of PDE4C, which is inhibited with a slightly lower potency. The half maximal inhibitory concentration (IC50) values for PDE4 subtype inhibition (Table 1) are in the range reported by others for the inhibition of truncated PDE4A–D versions [1].

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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.[3]

Studies on animals using Roflumilast have demonstrated that it decreases the build-up of neutrophils in bronchoalveolar lavage fluid following short-term tobacco smoke exposure in mice, rats, or guinea pigs; additionally, it eliminates the infiltration of inflammatory cells in the lung parenchyma of rats exposed to tobacco smoke for seven months [2]. In pIgR, rolumilast prevents the advancement of COPD?*? rats. 9-month-old WT or pIgR for these investigations?*? For three months, mice received oral gavage treatment with either 100 μg of Roflumilast (5 μg/g) or a vehicle (4% methylcellulose, 1.3% PEG400). Around the age of 12 months, the lungs were taken. When Roflumilast was administered to mice, minor airway wall remodeling did not advance as it did in vehicle-treated pIgR-/- animals. Surprisingly, pIgR aged 12 months who received rolumilast?*? Compared to 9-month-old pIgR, mice's emphysema index was lower.*? Roflumilast not only stops pulmonary emphysema from developing in this scenario, as demonstrated by the mice. appears to aid in the resolution of the emphysematous loss of lung parenchyma throughout the course of emphysema [4].

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In vivo, Roflumilast mitigates key COPD-related disease mechanisms such as tobacco smoke-induced lung inflammation, mucociliary malfunction, lung fibrotic and emphysematous remodelling, oxidative stress, pulmonary vascular remodelling and pulmonary hypertension. In vitro, roflumilast N-oxide has been demonstrated to affect the functions of many cell types, including neutrophils, monocytes/macrophages, CD4+ and CD8+ T-cells, endothelial cells, epithelial cells, smooth muscle cells and fibroblasts. These cellular effects are thought to be responsible for the beneficial effects of roflumilast on the disease mechanisms of COPD, which translate into reduced exacerbations and improved lung function. As a multicomponent disease, COPD requires a broad therapeutic approach that might be achieved by PDE4 inhibition. However, as a PDE4 inhibitor, roflumilast is not a direct bronchodilator. [1]

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Roflumilast blocks COPD progression in pIgR−/− mice [4]
Next, to investigate whether progressive small airway remodelling and emphysema in pIgR−/− mice occur in response to bacteria-induced inflammation, we used the anti-inflammatory drug roflumilast, which inhibits phosphodiesterase-4. Roflumilast is FDA approved for use in COPD patients and has been shown to reduce inflammation in murine models of COPD42,43,44,45. For these studies, 9-month-old WT or pIgR−/− mice were treated daily by oral gavage with 100 μg of roflumilast (5 μg g−1) or vehicle (4% methylcellulose, 1.3% PEG400) for 3 months and lungs were harvested at 12 months of age. Unlike pIgR−/− mice treated with vehicle, mice treated with roflumilast had no progression of small airway wall remodelling after starting treatment (Fig. 6a). Strikingly, 12-month-old pIgR−/− mice treated with roflumilast had reduced indices of emphysema compared with 9-month-old pIgR−/− mice, indicating that roflumilast not only blocks progression of emphysema in this model but apparently facilitates some resolution of the emphysematous destruction of lung parenchyma (Fig. 6b,c). Similar to mice housed in germ-free conditions, WT and pIgR−/− mice treated with roflumilast had very few neutrophils in the lung parenchyma (Fig. 6d) and macrophage numbers were equivalent to vehicle-treated WT mice (Fig. 6e). Consistent with decreased inflammation, roflumilast treatment resulted in reduced MMP-12 and NE in lungs of pIgR−/− mice (Fig. 6f and Supplementary Fig. 6). In addition, NF-κB activation and KC expression were reduced in lungs of roflumilast-treated pIgR−/− mice compared with vehicle-treated pIgR−/− mice (Fig. 6g,h and Supplementary Fig. 7). Together, these data indicate that persistent bacterial-derived inflammation propels COPD-like remodelling in pIgR−/− mice.

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Bodyweights of the HFD-fed rats significantly increased and were not ameliorated by Roflumilast treatment. Cystometry evidenced augmented frequency and non-void contractions in obese rats that were also prevented by roflumilast. These alterations were accompanied by a markedly increased expression of TNF-α, IL-6, IL-1β, and NF-κB in DSM of obese rats. Furthermore, roflumilast decreased expression of inflammatory factors in DSM. Conclusions: Oral treatment with roflumilast in rats fed an HFD restores normal bladder function and downregulates expression of inflammatory factors in the bladder. [5]

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Effect of v on healing bladder dysfunction in obese rats [5]
Oral roflumilast treatment for 4 weeks improved the bladder function, such as bladder capacity (0.54 ± 0.08 ml; N = 10), voiding volume (0.52 ± 0.08 ml; N = 10), voiding interval (2.8 ± 0.4 min; N = 10), and the frequency of NVCs (0.7 ± 0.4; N = 10) in obese rats, compared to rats from HFD + vehicle groups (N = 10; P < 0.05; Fig. 1). In contrast, maximum voiding pressure remained similar for roflumilast-treated HFD-fed rats (41.5 ± 6.8 cm H2O, N = 10; P > 0.05; Fig. 1a, d). Furthermore, the improved cystometric parameters detected after roflumilast treatment in obese rats were similar to the cystometric parameters in ND + vehicle rats (N = 10; P > 0.05; Fig. 1). Our present results indicated roflumilast induced an improvement in bladder function and voiding efficiency in obese rats.

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Roflumilast inhibits inflammatory response in obese rats [5]
To assess whether this PDE4 inhibitor is involved in the inhibition of the inflammatory response and consequent DO in obese rats, study animals were treated with oral roflumilast. Obese rats treated with roflumilast showed decreased expression of inflammatory cytokines (NF-κB 0.68 ± 0.06, TNF-α 0.41 ± 0.06, IL-6 0.39 ± 0.09, and IL-1β 0.36 ± 0.09) in bladder smooth muscle when compared to vehicle-treated obese rats, as demonstrated by the gray level (n = 16; P < 0.05; Fig. 2). Similarly, a qRT-PCR test confirmed that roflumilast treatment reduced the expression of inflammatory factor genes in obese rats (NF-κB 0.64 ± 0.08, TNF-α 0.39 ± 0.08, IL-6 0.37 ± 0.09, and IL-1β 0.41 ± 0.09; n = 16; P < 0.05; Fig. 3). Moreover, the reduced expression of inflammatory factor genes and proteins after roflumilast treatment in obese rats was similar to the expression profile in ND + vehicle rats (n = 16; P > 0.05; Figs. 2, 3). Therefore, PDE4 inhibitors may play a primary role in inhibiting the release of inflammatory mediators and the activation of immune cells.

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Roflumilast was first dissolved in an alkaline solution (0.1 N NaOH), titrated to pH 7.4 with 0.1 N HCl, and then diluted with normal saline.
The animals were adapted in the lab one week prior to the experiment and then randomized into five groups (ten rats in each group) that were reared in separate polycarbonate cages. Group (1) received vehicle only (PBS and 0.8% methylcellulose). Group (2) was given Roflumilast (1 mg/kg, P.O.) once daily for 7 consecutive days plus 0.5 ml of PBS solution (i.p.). Group (3) was injected with a single dose of CIS in a dose of 7 mg/kg, i. p (Rezvanfar et al., 2013). plus 0.8% methylcellulose (P.O). Group (4) was administered Roflumilast at a dose of 0.3 mg/kg, orally by oral gavage 30 min prior to CIS administration and continued for 7 consecutive days. Group (5) was administered Roflumilast at a dose of 1 mg/kg, orally by oral gavage 30 min prior to CIS administration and continued for 7 consecutive days. Doses and routes of Roflumilast administration were selected based on previously reported study [6].

Biochemical assays [6]
Assay of oxidative stress parameters [6]
The levels of malondialdehyde (MDA) (which is the marker of lipid peroxidation), NO, and GSH contents in a testicular homogenate were determined according to the methods described by Preuss et al. (1998), Grisham et al. (1996), and Griffith (1980), respectively. Testicular CAT activity was determined using a commercial assay kit according to the manufacturer's instructions.

Intracellular cAMP measurement [6]
Intracellular cAMP levels in testicular homogenates were measured using a cAMP enzyme immunoassay (ELISA) kit in accordance with the manufacturer's instructions.

Assay of cAMP-dependent protein kinase (PKA) and HO-1 activities [6]
For assay of PKA activity in testicular tissues, homogenate Abcam PKA Kinase Activity assay Kit was used. The kit is a sensitive, safe, non-radioactive ELISA assay providing a rapid and reliable method for quantitating the activity of PKA that utilizes a specific synthetic peptide as a substrate for PKA and a polyclonal antibody that identifies the phosphorylated form of the substrate. All procedures were done according to the manufacturer's instructions. For assay of HO-1 activity, samples of testicular homogenates were incubated in a mixture of heme (50 mmol/L), rat liver cytosol (5 mg/mL), MgCl2 (2 mmol/L), glucose-6-phosphate (2 mmol/L), glucose-6-phosphate dehydrogenase (1 unit), and nicotinamide adenine dinucleotide phosphate (NADPH) (0.8 mmol/L) in 0.5 mL PBS (pH 7.4) at 37 °C for 60 min. The reaction was stopped by immersing the tubes in ice for cooling. The bilirubin product was extracted, and its concentration was measured spectrophotometrically at 520 nm and was calculated by utilizing the extinction coefficient method (Abraham et al., 1988).

Assay of pro-inflammatory cytokines and apoptosis markers [6]
The levels of interleukin-1beta (IL-1β), tumor necrosis factor alpha (TNF-α) and apoptotic markers Bax and Bcl-2 in testicular tissue homogenate, were determined using specific rat ELISA kits purchased from R&D Systems. Moreover, the activity of Caspase-3 was measured by a colorimetric kit, following the standard manufacturer's instructions.

Determination of total proteins [6]
Lowry's method (Waterborg, 2009) for determining the total protein concentration within testicular tissues homogenate was used, and bovine plasma albumin was used as a standard.

Cytotoxicity study [6]
Cytotoxicity was assayed using Sulphorhodamine-B (SRB) method (Skehan et al., 1990). Cancer cells were seeded in 96 well flat-bottom plates for 24 h. After that, media was replaced with fresh media supplemented with appropriate drug concentrations. Different concentrations (0, 1, 5, 10, 25, and 50 mg/mL) of the tested drugs; CIS, v were added to the cell monolayer for 48 h at 37 °C. Triplicate wells were prepared for each dose level for the determination of IC50 values (the concentration at which 50% of cell growth is inhibited) for each drug. In another experiment, a combination of IC50 of CIS (3.9 mg/mL) and IC50 of Roflumilast (2.3 mg/mL) was added to the cells to determine the surviving fraction% and inhibiting fraction%. Roflumilast and other compounds were initially dissolved in dimethyl sulfoxide (DMSO) and further diluted to the working solution in the culture medium. The final concentration of DMSO in all treatments did not exceed 0.1% (v/v) in the medium, which had no discernible effect on cell killing. After treatment, cells were fixed with 10% trichloroacetic acid for 1 h at 4 °C. Wells then were washed with water and then stained with 0.4% SRB in 1% acetic acid for 30 min at 25 °C. The dye was solubilized with 10 mM trizma® base (pH 10.5). The resulted color change was measured spectrophotometrically at 564 nm. The IC50 value was calculated from the plotted survival fraction curve of the cells from the relation between surviving fraction and drug concentration.

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Quantitative RT-PCR [6]
The effect of Roflumilast on the CIS-induced changes in the gene expression of the signaling transcription factors and apoptotic markers in the rat testicles and the PC3 cancer cell line was determined using quantitative RT-PCR, with β-actin as the reference gene. For the in vivo study, total RNA from the testicular tissues was prepared using a TRIzol isolation kit, was purified using a RNeasy purification kit, and was assayed spectrophotometrically at 260 nm. For the in vitro cytotoxicity study, the PC3 prostate cancer cell line was plated as explained above. Twenty-four hours later, the medium was removed and replaced with a fresh medium containing one of the following: IC50 of cisplatin (3.9 mg/mL), IC50 of Roflumilast (4.7 mg/mL), or a combination of cisplatin with Roflumilast for 48 h at 37 °C. Consequently, the cells were collected, washed with ice-cold PBS, and lysed.

Roflumilast administration [4]
For studies using roflumilast, 200 μl of 0.5 mg ml−1 suspension of Roflumilast or vehicle (4% methylcellulose, 1.3% PEG400 and ∼5 μg drug per mg animal weight) was administered by oral gavage once daily, 5 days a week for the duration of treatment. The roflumilast suspension was freshly prepared each week and stored at 4 °C.

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Diet-induced obesity and study treatment [5]
For 12 weeks, study animals were housed three per cage on a 12-h light–dark cycle, and either normal diet (ND) (fat: 5%; protein: 20%; carbohydrate: 75%) or HFD (fat: 30%; protein: 14%; carbohydrate: 56%) that induces obesity as previously described [17, 18]. Study animals were divided into three groups (N = 30 in each group): (1) vehicle-treated ND-fed (ND + vehicle) rats (normal diet for 8 weeks before receiving the vehicle); (2) vehicle-treated HFD-fed (HFD + vehicle) rats (HFD for 8 weeks before receiving the vehicle); and (3) Roflumilast-treated HFD-fed (HFD + roflumilast) rats (HFD for 8 weeks before receiving roflumilast). Roflumilast (5 mg/kg/day) or vehicle (sterile water used as solvent for roflumilast) was administered orally by gavage during the last 4 weeks of HFD or ND feeding. All rats were weighed at 12 weeks, and urodynamic studies were conducted in ten rats of each group. Study animals were then killed in a carbon dioxide tank prior to collection of bladder specimens; the bladder mucosa was separated under microscopy, and the DSM tissue was preserved in liquid nitrogen.

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Methods: In this 12-week study, 90 female Sprague-Dawley rats were divided into three groups: (1) vehicle-treated normal diet (ND)-fed rats; (2) vehicle-treated high-fat diet (HFD)-fed rats; and (3) Roflumilast-treated HFD-fed rats. Oral roflumilast (5 mg/kg/day) was administered during the last 4 weeks of HFD feeding in the test group. At 12 weeks, a urodynamic study was performed in ten rats of each group. Bladder tissue was extracted, the bladder mucosa was separated under microscopy, and bladder detrusor smooth muscle (DSM) expression of TNF-α, interleukin (IL)-6, IL-1β, and nuclear factor kappa B (NF-κB) were analyzed using Western blotting and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) [5].

Absorption, Distribution and Excretion
After a 500mcg dose, the bioavailability of Roflumilast is about 80%. In the fasted state, maximum plasma concentrations are reached in 0.5 to 2 hours, while in the fed state, Cmax is reduced by 40%, Tmax is increased by one hour, and total absorption is unchanged. Applied topically, the mean systemic exposure for roflumilast and its N-oxide metabolite in adults was 72.7 ± 53.1 and 628 ± 648 h∙ng/mL, respectively. The mean systemic exposure for roflumilast and its N-oxide metabolite in adolescents was 25.1 ± 24.0 and 140 ± 179 h∙ng/mL, respectively.
Roflumilast is excreted 70% in the urine as roflumilast N-oxide.
Following a single oral dose of 500 mcg, the volume of distribution of roflumilast is approximately 2.9 L/kg.
Plasma clearance of roflumilast following short-term intravenous infusion is approximately 9.6 L/h.

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Metabolism / Metabolites
Roflumilast is metabolized to roflumilast N-oxide, the active metabolite of roflumilast in humans, by CYP3A4 and CYP1A2. The N-oxide metabolite is less potent than its parent drug in regards to PDE4 inhibition, but its plasma AUC is approximately 10-fold greater.

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Biological Half-Life
Following oral administration, the plasma half-lives of roflumilast and roflumilast N-oxide are 17 hours and 30 hours, respectively.

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Roflumilast is rapidly metabolised to its N-oxide at the dichloropyridyl moiety by cytochrome P450 (CYP) 3A4 and CYP1A2 enzymes (Fig. 1A). As indicated in Table 1, roflumilast N-oxide is only 2–3-fold less potent than the parent compound with respect to PDE4 inhibition, maintains high selectivity to other PDE isoenzymes and shows no selectivity for PDE4 subtypes. In man, this metabolite is estimated to account for about 90% of the overall PDE4 inhibition and 10% is attributed to the roflumilast parent. On repeated oral dosing with roflumilast 500 μg once daily in healthy subjects, the free drug concentration of roflumilast N-oxide in plasma over 24 h was estimated to be about 1–2 nM by considering the plasma protein binding of roflumilast N-oxide of approximately 97% (Fig. 1B). Although it is well-known that tobacco smoking increases CYP1A2, it was found that smoking has a minor influence on the pharmacokinetic profile of roflumilast. Before information was available for roflumilast, intriguing clinical results were reported in COPD and asthma for cilomilast, an extensively characterised PDE4 inhibitor. However, no further development of cilomilast has been reported recently. Unlike roflumilast and its N-oxide, cilomilast shows some subtype selectivity for PDE4D (Table 1). This may be a disadvantage in terms of adverse events, as PDE4D may be linked to emesis and/or cardiovascular side-effects in patients at risk of heart failure [1].

Hepatotoxicity
In preregistration studies, roflumilast was not associated with serum enzyme elevations or with episodes of clinically apparent liver injury. Since approval of roflumilast, there have been no published reports of hepatotoxicity, and the product label does not mention liver injury as an adverse event.
Likelihood score: E (unlikely cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the use of roflumilast in nursing mothers. The drug and its metabolite are more than 97% bound to plasma proteins, so amounts in milk are likely to be very low. However, the manufacturer and some experts recommend that the oral drug should not be used by women who are nursing.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Plasma protein binding of roflumilast and its N-oxide metabolite is approximately 99% and 97%, respectively.

[1]. The preclinical pharmacology of roflumilast--a selective, oral phosphodiesterase 4 inhibitor in development for chronic obstructive pulmonary disease. Pulm Pharmacol Ther. 2010 Aug;23(4):235-56.

[2]. Update on roflumilast, a phosphodiesterase 4 inhibitor for the treatment of chronic obstructive pulmonary disease. Br J Pharmacol. 2011 May;163(1):53-67.

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

[4]. Airway bacteria drive a progressive COPD-like phenotype in mice with polymeric immunoglobulin receptor deficiency. Nat Commun. 2016 Apr 5;7:11240.

[5]. Treatment of obesity-associated overactive bladder by the phosphodiesterase type-4 inhibitor roflumilast. Int Urol Nephrol. 2017 Oct;49(10):1723-1730.

[6]. Roflumilast protects from cisplatin-induced testicular toxicity in male rats and enhances its cytotoxicity in prostate cancer cell line. Role of NF-κB-p65, cAMP/PKA and Nrf2/HO-1, NQO1 signaling. Food Chem Toxicol. 2021 May:151:112133.

Pharmacodynamics
Roflumilast and its active metabolite, roflumilast N-oxide, increase cyclic adenosine-3′, 5′-monophosphate (cAMP) in affected cells by inhibiting PDE4. They are highly selective for PDE4 and are effectively inactive against PDEs 1, 2, 3, 5, and 7.

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Roflumilast is a benzamide obtained by formal condensation of the carboxy group of 3-(cyclopropylmethoxy)-4-(difluoromethoxy)benzoic acid with the amino group of 3,5-dichloropyridin-4-amine. Used for treatment of bronchial asthma and chronic obstructive pulmonary disease. It has a role as a phosphodiesterase IV inhibitor and an anti-asthmatic drug. It is a member of benzamides, a chloropyridine, an aromatic ether, an organofluorine compound and a member of cyclopropanes.
Roflumilast is a highly selective phosphodiesterase-4 (PDE4) inhibitor. PDE4 is a major cyclic-3',5′-adenosinemonophosphate (cyclic AMP, cAMP)-metabolizing enzyme expressed on nearly all immune and pro-inflammatory cells, in addition to structural cells like those of the smooth muscle or epithelium. The resultant increase in intracellular cAMP induced by roflumilast's inhibition of PDE4 is thought to mediate its disease-modifying effects, although its precise mechanism of action has yet to be elucidated. The oral formulation of roflumilast is indicated to manage chronic obstructive pulmonary disease. It was first approved by the EMA in July 2010, and by the FDA in January 2018. Roflumilast topical cream is indicated to treat plaque psoriasis. The cream formulation was first approved by the FDA in July 2022 and by Health Canada in April 2023. On December 15, 2023, the FDA approved a new topical foam formulation of roflumilast for the treatment of seborrheic dermatitis in patients aged 9 years and older.

Roflumilast is a Phosphodiesterase 4 Inhibitor. The mechanism of action of roflumilast is as a Phosphodiesterase 4 Inhibitor.
Roflumilast is a selective inhibitor of phosphodiesterase-4 (PDE-4) that has unique antiinflammatory activity and is used to treat and prevent exacerbations of chronic obstructive pulmonary disease (COPD). Roflumilast has not been linked to significant serum enzyme elevations during therapy or to instances of clinically apparent acute liver injury.
Roflumilast is an orally available, long-acting inhibitor of phosphodiesterase (PDE) type 4 (PDE4), with anti-inflammatory and potential antineoplastic activities. Upon administration, roflumilast and its active metabolite roflumilast N-oxide selectively and competitively bind to and inhibit PDE4, which leads to an increase of both intracellular levels of cyclic-3',5'-adenosine monophosphate (cAMP) and cAMP-mediated signaling. cAMP prevents phosphorylation of spleen tyrosine kinase (SYK) and abrogates activation of the PI3K/AKT/mTOR signaling pathway, which may result in the induction of apoptosis. PDE4, a member of the PDE superfamily that hydrolyses cAMP and 3',5'-cyclic guanosine monophosphate (cGMP) to their inactive 5' monophosphates, is upregulated in a variety of cancers and may contribute to chemoresistance; it also plays a key role in inflammation, especially in inflammatory airway diseases.
Roflumilast is a small molecule drug with a maximum clinical trial phase of IV (across all indications) that was first approved in 2010 and has 3 approved and 19 investigational indications.

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After more than two decades of research into phosphodiesterase 4 (PDE4) inhibitors, Roflumilast (3-cyclopropylmethoxy-4-difluoromethoxy-N-[3,5-di-chloropyrid-4-yl]-benzamide) may become the first agent in this class to be approved for patient treatment worldwide. Within the PDE family of 11 known isoenzymes, roflumilast is selective for PDE4, showing balanced selectivity for subtypes A–D, and is of high subnanomolar potency. The active principle of roflumilast in man is its dichloropyridyl N-oxide metabolite, which has similar potency as a PDE4 inhibitor as the parent compound. The long half-life and high potency of this metabolite allows for once-daily, oral administration of a single, 500-μg tablet of roflumilast.
The molecular mode of action of roflumilast – PDE4 inhibition and subsequent enhancement of cAMP levels – is well established. To further understand its functional mode of action in chronic obstructive pulmonary disease (COPD), for which roflumilast is being developed, a series of in vitro and in vivo preclinical studies has been performed.
COPD is a progressive, devastating condition of the lung associated with an abnormal inflammatory response to noxious particles and gases, particularly tobacco smoke. In addition, according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD), significant extrapulmonary effects, including comorbidities, may add to the severity of the disease in individual patients, and which may be addressed preferentially by orally administered remedies. COPD shows an increasing prevalence and mortality, and its treatment remains a high, unmet medical need.
In vivo, Roflumilast mitigates key COPD-related disease mechanisms such as tobacco smoke-induced lung inflammation, mucociliary malfunction, lung fibrotic and emphysematous remodelling, oxidative stress, pulmonary vascular remodelling and pulmonary hypertension. In vitro, roflumilast N-oxide has been demonstrated to affect the functions of many cell types, including neutrophils, monocytes/macrophages, CD4+ and CD8+ T-cells, endothelial cells, epithelial cells, smooth muscle cells and fibroblasts. These cellular effects are thought to be responsible for the beneficial effects of roflumilast on the disease mechanisms of COPD, which translate into reduced exacerbations and improved lung function. As a multicomponent disease, COPD requires a broad therapeutic approach that might be achieved by PDE4 inhibition. However, as a PDE4 inhibitor, Roflumilast is not a direct bronchodilator.
In summary, roflumilast may be the first-in-class PDE4 inhibitor for COPD therapy. In addition to being a non-steroid, anti-inflammatory drug designed to target pulmonary inflammation, the preclinical pharmacology described in this review points to a broad functional mode of action of roflumilast that putatively addresses additional COPD mechanisms. This enables roflumilast to offer effective, oral maintenance treatment for COPD, with an acceptable tolerability profile and the potential to favourably affect the extrapulmonary effects of the disease. [1]

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Phosphodiesterase 4 (PDE4) is a member of the PDE enzyme superfamily that inactivates cyclic adenosine monophosphate and cyclic guanosine monophosphate, and is the main PDE isoenzyme occurring in cells involved in inflammatory airway disease such as chronic obstructive pulmonary disease (COPD). COPD is a preventable and treatable disease and is characterized by airflow obstruction that is not fully reversible. Chronic progressive symptoms, particularly dyspnoea, chronic bronchitis and impaired overall health are worse in those who have frequent, acute episodes of symptom exacerbation. Although several experimental PDE4 inhibitors are in clinical development, Roflumilast, a highly selective PDE4 inhibitor, is the first in its class to be licensed, and has recently been approved in several countries for oral, once-daily treatment of severe COPD. Clinical trials have demonstrated that roflumilast improves lung function and reduces exacerbation frequency in COPD. Furthermore, its unique mode of action may offer the potential to target the inflammatory processes underlying COPD. Roflumilast is effective when used concomitantly with all forms of bronchodilator and even in patients treated with inhaled corticosteroids. Roflumilast thus represents an important addition to current therapeutic options for COPD patients with chronic bronchitis, including those who remain symptomatic despite treatment. This article reviews the current status of PDE4 inhibitors, focusing on the pharmacokinetics, efficacy and safety of roflumilast. In particular, it provides an overview of the effects of roflumilast on lung function and exacerbations, glucose homoeostasis and weight loss, and the concomitant use of long-acting beta(2)-adrenergic receptor agonists and short-acting muscarinic receptor antagonists. [2]

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Mechanisms driving persistent airway inflammation in chronic obstructive pulmonary disease (COPD) are incompletely understood. As secretory immunoglobulin A (SIgA) deficiency in small airways has been reported in COPD patients, we hypothesized that immunobarrier dysfunction resulting from reduced SIgA contributes to chronic airway inflammation and disease progression. Here we show that polymeric immunoglobulin receptor-deficient (pIgR(-/-)) mice, which lack SIgA, spontaneously develop COPD-like pathology as they age. Progressive airway wall remodelling and emphysema in pIgR(-/-) mice are associated with an altered lung microbiome, bacterial invasion of the airway epithelium, NF-κB activation, leukocyte infiltration and increased expression of matrix metalloproteinase-12 and neutrophil elastase. Re-derivation of pIgR(-/-) mice in germ-free conditions or treatment with the anti-inflammatory phosphodiesterase-4 inhibitor Roflumilast prevents COPD-like lung inflammation and remodelling. These findings show that pIgR/SIgA deficiency in the airways leads to persistent activation of innate immune responses to resident lung microbiota, driving progressive small airway remodelling and emphysema. [4]

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Purpose
To prove that phosphodiesterase type-4 inhibitors could potentially treat obesity-associated overactive bladder through modulation of the systemic inflammatory response.

Methods
In this 12-week study, 90 female Sprague–Dawley rats were divided into three groups: (1) vehicle-treated normal diet (ND)-fed rats; (2) vehicle-treated high-fat diet (HFD)-fed rats; and (3) Roflumilast-treated HFD-fed rats. Oral roflumilast (5 mg/kg/day) was administered during the last 4 weeks of HFD feeding in the test group. At 12 weeks, a urodynamic study was performed in ten rats of each group. Bladder tissue was extracted, the bladder mucosa was separated under microscopy, and bladder detrusor smooth muscle (DSM) expression of TNF-α, interleukin (IL)-6, IL-1β, and nuclear factor kappa B (NF-κB) were analyzed using Western blotting and quantitative reverse transcription-polymerase chain reaction (qRT-PCR).

Results
Bodyweights of the HFD-fed rats significantly increased and were not ameliorated by Roflumilast treatment. Cystometry evidenced augmented frequency and non-void contractions in obese rats that were also prevented by roflumilast. These alterations were accompanied by a markedly increased expression of TNF-α, IL-6, IL-1β, and NF-κB in DSM of obese rats. Furthermore, roflumilast decreased expression of inflammatory factors in DSM.

Conclusions
Oral treatment with Roflumilast in rats fed an HFD restores normal bladder function and downregulates expression of inflammatory factors in the bladder.[5]

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Cisplatin (CIS)-induced testicular injury is a major obstacle in its application as antineoplastic agent. In this study, we investigated the protective effect and mechanism of Roflumilast (ROF), a PDE4 inhibitor, against CIS-induced testicular toxicity in rats. Besides, the cytotoxic effect of CIS, with and without ROF, was evaluated on PC3 cell line. ROF reversed CIS-induced abnormalities in sperm characteristics, normalized serum testosterone level, and ameliorated CIS-induced alterations in testicular and epidydimal weights and restored normal testicular structure. Moreover, ROF increased intracellular cAMP level, PKA and HO-1 activities and Nrf2, NQO-1 and HO-1 gene expression, improved testicular oxidative stress parameters (TBARS, NO, GSH levels, and CAT activity) and inflammatory mediators (IL-1β and TNF-α, and NF-κβ p65gene expression) and reduced the proapoptotic proteins, caspase-3, Bax and increased Bcl-2. Lastly, in vitro analyses showed that ROF augmented the anticancer efficacy of CIS and enhanced the increase in gene expression of Nrf2, HO-1, and NQO-1 and the inhibition of gene expression of NF-κβ p65 induced by CIS and enhanced its apoptotic effect in PC3 cells. Conclusively, PDE4 inhibition with induction of Nrf2/HO-1, NQO-1 is a potential therapeutic approach to protect male reproductive system from the detrimental effects with augmenting, the antineoplastic effect of CIS. [6]

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The results of the current study provide evidence that ROF as a PDE4 inhibitor has a protective effect against CIS-induced male reproductive toxicity. In addition, it provides evidence that inhibition of PDE4 by ROF stimulates the cAMP/Nrf2/HO-1, NQO-1 signaling cascade, which plays a vital role in mitigating the oxidative damage and inflammatory response and attenuated testicular injury induced by CIS in rats. In addition, ROF alone or in combination with CIS triggers prostatic cancer PC3 cell apoptosis by increasing protein and gene expressions of caspase-3, Bax, and Bax/Bcl-2 ratio and decreasing both protein and gene expression of Bcl-2, the effect that can be attributed to stimulation of Nrf2/HO-1 expression and down-regulation of NF-κβ p65 gene expression. Hence, the current study provides a new promising strategy that augments the anticancer effect of CIS and further alleviates its testicular toxicity by its combination with ROF. Further studies in the future are needed to further evaluate the possible clinical application of this combination in cancer chemotherapy.[6]

C17H14CL2F2N2O3

403.2075

402.034

C, 50.64; H, 3.50; Cl, 17.58; F, 9.42; N, 6.95; O, 11.90

162401-32-3

Roflumilast N-oxide;292135-78-5;Roflumilast-d4 N-Oxide;1794760-31-8;Roflumilast Impurity E;1391052-76-8;Roflumilast-d4;1398065-69-4;Roflumilast-d3;1189992-00-4

449193

White to off-white solid powder

1.5±0.1 g/cm3

430.6±45.0 °C at 760 mmHg

158°C

214.2±28.7 °C

0.0±1.0 mmHg at 25°C

1.604

4.84

1

6

7

26

475

0

C1CC1COC2=C(C=CC(=C2)C(=O)NC3=C(C=NC=C3Cl)Cl)OC(F)F

MNDBXUUTURYVHR-UHFFFAOYSA-N

InChI=1S/C17H14Cl2F2N2O3/c18-11-6-22-7-12(19)15(11)23-16(24)10-3-4-13(26-17(20)21)14(5-10)25-8-9-1-2-9/h3-7,9,17H,1-2,8H2,(H,22,23,24)

3-(cyclopropylmethoxy)-N-(3,5-dichloropyridin-4-yl)-4-(difluoromethoxy)benzamide

Daliresp; BY217; BY-217; Roflumilast; 162401-32-3; DAXAS; 3-(CYCLOPROPYLMETHOXY)-N-(3,5-DICHLOROPYRIDIN-4-YL)-4-(DIFLUOROMETHOXY)BENZAMIDE; Daliresp; BYK20869; Benzamide, 3-(cyclopropylmethoxy)-N-(3,5-dichloro-4-pyridinyl)-4-(difluoromethoxy)-; B 9302-107;BYK 20869;B-9302-107;APTA 2217, B9302-107, BY 217, BYK-20869; BYK20869; Daxas;

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 (6.20 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 2: 30% PEG400+0.5% Tween80+5% propylene glycol:30 mg/mL

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