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
Following an oral dose of 500 mcg of roflumilast, its bioavailability is approximately 80%. In the fasting state, peak plasma concentrations are reached within 0.5 to 2 hours; in the fed state, peak concentrations decrease by 40%, and the time to peak concentration is prolonged by 1 hour, while total absorption remains unchanged. For topical application, the mean systemic exposures of roflumilast and its N-oxide metabolites in adults are 72.7 ± 53.1 h∙ng/mL and 628 ± 648 h∙ng/mL, respectively. In adolescents, the mean systemic exposures of roflumilast and its N-oxide metabolites are 25.1 ± 24.0 h∙ng/mL and 140 ± 179 h∙ng/mL, respectively. 70% of roflumilast is excreted 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.
Following short-term intravenous infusion of roflumilast, its plasma clearance is approximately 9.6 L/h.
Metabolism/Metabolites
Roflumilast is metabolized by CYP3A4 and CYP1A2 to roflumilast N-oxide, which is the metabolite that makes roflumilast active in the human body. The roflumilast N-oxide metabolite is less potent than its parent drug in inhibiting PDE4, but its plasma AUC is approximately 10 times that of the parent drug.
Biological Half-Life
After oral administration, the plasma half-lives of roflumilast and its N-oxide are 17 hours and 30 hours, respectively.
Roflumilast
In the dichloropyridine fraction, it is rapidly metabolized to N-oxide by cytochrome P450 (CYP) 3A4 and CYP1A2 enzymes (Figure 1A). As shown in Table 1, roflumilast N-oxide exhibits only 2-3 times lower PDE4 inhibition than its parent compound, maintains high selectivity for other PDE isoenzymes, and shows no selectivity for PDE4 subtypes. In humans, this metabolite is estimated to account for approximately 90% of the total PDE4 inhibition, with the remaining 10% attributed to the roflumilast parent drug. In healthy subjects, after a once-daily oral administration of 500 μg roflumilast, the estimated free roflumilast N-oxide concentration in plasma over 24 hours was approximately 1-2 nM, based on an estimated 97% plasma protein binding rate (Figure 1B). Although smoking is known to increase CYP1A2 expression, studies have found that smoking has minimal impact on the pharmacokinetic characteristics of roflumilast. Prior to the publication of information on roflumilast, cilomylast, a widely studied PDE4 inhibitor, demonstrated remarkable clinical efficacy in the treatment of chronic obstructive pulmonary disease (COPD) and asthma. However, no further development progress of cilomylast has been reported recently. Unlike roflumilast and its N-oxide, silloster exhibits some selectivity for PDE4D subtypes (Table 1). This may be a drawback in terms of adverse events, as PDE4D may be associated with vomiting and/or cardiovascular side effects in patients at risk of heart failure [1].

Hepatotoxicity
In pre-registration studies, roflumilast was not associated with elevated serum enzymes or clinically significant liver injury. Since roflumilast's approval, there have been no published reports of hepatotoxicity, and liver injury is not listed as an adverse event in the product information leaflet. Likelihood score: E (unlikely to cause clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no information on the use of roflumilast in breastfeeding women. This drug and its metabolites bind to plasma proteins at a rate exceeding 97%, therefore the concentration in breast milk may be very low. However, the manufacturer and some experts recommend that breastfeeding women should not take this oral medication. ◉ Effects on Breastfed Infants As of the revision date, no relevant published information was found. ◉ Effects on Lactation and Breast Milk As of the revision date, no relevant published information was found.

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Protein Binding
Roflumilast and its N-oxide metabolites have plasma protein binding rates of 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 monophosphate (cAMP) levels in affected cells by inhibiting PDE4. They are highly selective for PDE4 and have little activity against PDE1, 2, 3, 5, and 7. Roflumilast is a benzamide formed by the condensation of the carboxyl group of 3-(cyclopropylmethoxy)-4-(difluoromethoxy)benzoic acid with the amino group of 3,5-dichloropyridin-4-amine. It is used to treat bronchial asthma and chronic obstructive pulmonary disease. It is a phosphodiesterase IV inhibitor and an anti-asthmatic drug. Roflumilast belongs to the benzamide, chloropyridine, aromatic ether, organofluorine, and cyclopropane classes. Roflumilast is a highly selective phosphodiesterase-4 (PDE4) inhibitor. PDE4 is a major cyclic adenosine monophosphate (cAMP) metabolic enzyme, expressed on almost all immune cells, pro-inflammatory cells, and structural cells such as smooth muscle and epithelium. Roflumilast induces an increase in intracellular cAMP levels by inhibiting PDE4, which is believed to be its mechanism of action in improving disease efficacy, but its exact mechanism of action is not fully elucidated. Roflumilast oral formulations are indicated for the treatment of chronic obstructive pulmonary disease (COPD). Roflumilast was first approved by the European Medicines Agency (EMA) in July 2010 and by the U.S. Food and Drug Administration (FDA) in January 2018. Roflumilast cream is indicated for the treatment of plaque psoriasis. This 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 roflumilast topical foam formulation for the treatment of seborrheic dermatitis in patients 9 years of age and older. Roflumilast is a phosphodiesterase 4 (PDE4) inhibitor. Roflumilast's mechanism of action is as a phosphodiesterase-4 (PDE-4) inhibitor. Roflumilast is a selective PDE-4 inhibitor with unique anti-inflammatory activity, used to treat and prevent acute exacerbations of chronic obstructive pulmonary disease (COPD). Roflumilast has not caused significant increases in serum enzymes or clinically significant acute liver injury during treatment. Roflumilast is an orally administered, long-acting PDE4 inhibitor with anti-inflammatory and potential antitumor activities. After administration, roflumilast and its active metabolite, roflumilast N-oxide, selectively and competitively bind to and inhibit PDE4, leading to increased intracellular cyclic adenosine monophosphate (cAMP) levels and activation of cAMP-mediated signaling pathways. cAMP inhibits phosphorylation of spleen tyrosine kinase (SYK) and blocks activation of the PI3K/AKT/mTOR signaling pathway, which may induce apoptosis. PDE4 is a member of the PDE superfamily that hydrolyzes cAMP and 3',5'-cyclic guanosine monophosphate (cGMP) into inactive 5'-monophosphate. PDE4 is upregulated in various cancers, potentially leading to chemotherapy resistance; it also plays a crucial role in inflammation, particularly in inflammatory airway diseases. Roflumilast is a small molecule drug, with clinical trials up to Phase IV (covering all indications), first approved in 2010, and currently has 3 approved indications and 19 investigational indications. After more than two decades of research on phosphodiesterase 4 (PDE4) inhibitors, Roflumilast (3-cyclopropylmethoxy-4-difluoromethoxy-N-[3,5-dichloropyridin-4-yl]-benzamide) is expected to become the first drug in this class to be approved for global patient treatment. Of the 11 known PDE isoenzymes, roflumilast exhibits selectivity for PDE4 and balanced selectivity for AD subtypes, along with extremely high subnanomolar potency. The active component of roflumilast in the human body is its metabolite, dichloropyridine N-oxide, which has similar potency as a PDE4 inhibitor to the parent compound. Due to the long half-life and high potency of this metabolite, 500 mcg roflumilast tablets can be taken orally once daily. The molecular mechanism of action of roflumilast—inhibition of PDE4 followed by an increase in cAMP levels—has been well elucidated. To further understand the mechanism of action of roflumilast in chronic obstructive pulmonary disease (COPD), which is currently being developed for the treatment of COPD, we have conducted a series of in vitro and in vivo preclinical studies. COPD is a progressive, destructive lung disease associated with an abnormal inflammatory response to harmful particles and gases, particularly tobacco smoke. Furthermore, according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD), significant extrapulmonary effects (including comorbidities) may exacerbate patients' conditions, and oral medications can prioritize addressing these effects. The prevalence and mortality of COPD are continuously rising, and its treatment remains a significant unmet medical need. In vivo, roflumilast can alleviate key COPD-related disease mechanisms, such as lung inflammation caused by tobacco smoke, mucociliary dysfunction, pulmonary fibrosis and emphysematous remodeling, oxidative stress, pulmonary vascular remodeling, and pulmonary hypertension. In vitro studies have shown that roflumilast N-oxide can affect the function of multiple cell types, including neutrophils, monocytes/macrophages, CD4+ and CD8+ T cells, endothelial cells, epithelial cells, smooth muscle cells, and fibroblasts. These cellular effects are believed to be the reason why roflumilast has a beneficial effect on the disease mechanisms of chronic obstructive pulmonary disease (COPD), thereby reducing acute exacerbations and improving lung function. COPD is a multi-component disease requiring a broad range of treatment approaches, and PDE4 inhibitors may help achieve this goal. However, as a PDE4 inhibitor, roflumilast is not a direct bronchodilator. In summary, roflumilast may be the first PDE4 inhibitor for the treatment of COPD. In addition to being a nonsteroidal anti-inflammatory drug designed to target lung inflammation, the preclinical pharmacological studies described in this review have shown that roflumilast has a broad range of functional mechanisms of action that may target other mechanisms of chronic obstructive pulmonary disease (COPD). This makes roflumilast an effective oral maintenance therapy for COPD with acceptable tolerability and the potential to have a positive effect on the extrapulmonary effects of the disease. [1] Phosphodiesterase 4 (PDE4) is a member of the phosphodiesterase superfamily that inactivates cyclic adenosine monophosphate and cyclic guanosine monophosphate and is the main PDE isoenzyme in cells involved in inflammatory airway diseases such as chronic obstructive pulmonary disease (COPD). COPD is a preventable and treatable disease characterized by airflow obstruction that is not completely reversible. Chronic progressive symptoms, especially dyspnea, chronic bronchitis and impaired overall health, are more severe in patients with frequent acute exacerbations. Although several experimental PDE4 inhibitors are in clinical development, roflumilast, a highly selective PDE4 inhibitor, is the first drug of its kind to be approved for marketing and has been approved in several countries for the treatment of severe chronic obstructive pulmonary disease (COPD) for once-daily oral administration. Clinical trials have shown that roflumilast can improve lung function and reduce the frequency of acute exacerbations in COPD patients. Furthermore, its unique mechanism of action may help target the underlying inflammatory process of COPD. Roflumilast is effective when used in combination with all types of bronchodilators, even in patients receiving inhaled corticosteroids. Therefore, roflumilast provides an important treatment option for patients with COPD and chronic bronchitis, including those who still have symptoms despite treatment. This article reviews the current status of PDE4 inhibitors, focusing on the pharmacokinetics, efficacy, and safety of roflumilast. The article specifically summarizes the effects of roflumilast on lung function and acute exacerbations, glucose homeostasis, and weight loss, as well as its use in combination with long-acting β2-adrenergic receptor agonists and short-acting muscarinic receptor antagonists. [2] The driving mechanisms of persistent airway inflammation in chronic obstructive pulmonary disease (COPD) are not fully understood. Given the reported deficiency of secretory immunoglobulin A (SIgA) in the small airways of COPD patients, we hypothesized that impaired immune barrier function due to SIgA reduction exacerbates chronic airway inflammation and promotes disease progression. This study demonstrates that SIgA-deficient polymerase receptor-deficient (pIgR^-/-) mice spontaneously develop COPD-like pathological changes with age. Progressive airway wall remodeling and emphysema in pIgR(-/-) mice are associated with altered lung microbiota, bacterial invasion of the airway epithelium, NF-κB activation, leukocyte infiltration, and increased expression of matrix metalloproteinase-12 and neutrophil elastase. Rebreeding pIgR(-/-) mice under sterile conditions or treatment with the anti-inflammatory phosphodiesterase-4 inhibitor roflumilast can prevent COPD-like lung inflammation and remodeling. These findings suggest that the lack of pIgR/SIgA in the airways leads to the sustained activation of the innate immune response to the lung’s native microbiota, thereby driving progressive small airway remodeling and emphysema. [4]

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Objective
To demonstrate that phosphodiesterase type 4 inhibitors can treat obesity-related overactive bladder by modulating the systemic inflammatory response.
Methods
This study lasted 12 weeks and 90 female Sprague-Dawley rats were divided into three groups: (1) normal diet (ND) group (carrier control group); (2) high-fat diet (HFD) group (carrier control group); (3) high-fat diet (HFD) group (roflumilast treatment group). The experimental groups were given oral roflumilast (5 mg/kg/day) for the last 4 weeks of high-fat diet feeding. After 12 weeks, urodynamics was performed on 10 rats in each group. Bladder tissue was extracted, and the bladder mucosa was separated under a microscope. The expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β, and nuclear factor-κB (NF-κB) in the detrusor smooth muscle (DSM) of the bladder was analyzed using Western blotting and quantitative reverse transcription-polymerase chain reaction (qRT-PCR).
Results
Rats fed a high-fat diet showed a significant increase in body weight, which was not alleviated by roflumilast treatment. Cystometry revealed increased urination frequency and non-voiding contractions in obese rats, which roflumilast inhibited. These changes were accompanied by a significant increase in the expression of TNF-α, IL-6, IL-1β, and NF-κB in the DSM of obese rats. Furthermore, roflumilast reduced the expression of inflammatory factors in deep bladder tissue.
Conclusion
In rats fed a high-fat diet, oral administration of roflumilast restored normal bladder function and downregulated the expression of inflammatory factors in the bladder. [5]

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Cisplatin (CIS)-induced testicular damage is a major obstacle to its use as an antitumor drug. This study investigated the protective effect and mechanism of the PDE4 inhibitor roflumilast (ROF) against cisplatin-induced testicular toxicity in rats. In addition, the cytotoxic effects of cisplatin on the PC3 cell line in the presence and absence of roflumilast were evaluated. Roflumilast reversed the abnormal sperm characteristics induced by cisplatin, restored serum testosterone levels to normal, improved the changes in testicular and epididymal weight induced by cisplatin, and restored normal testicular structure. In addition, ROF increased intracellular cAMP levels, 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 p65 gene expression), and decreased the expression of pro-apoptotic proteins caspase-3 and Bax, while increasing Bcl-2 expression. Finally, in vitro analysis showed that ROF could enhance the anticancer efficacy of CIS, enhance the increase in CIS-induced Nrf2, HO-1 and NQO-1 gene expression and the inhibition of NF-κβ p65 gene expression, and enhance its apoptosis effect in PC3 cells. In summary, PDE4 inhibition and induction of Nrf2/HO-1 and NQO-1 expression is a potential therapeutic approach that can protect the male reproductive system from the harmful effects of CIS and enhance the antitumor effect of CIS. [6]

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The results of this study indicate that ROF, as a PDE4 inhibitor, has a protective effect against CIS-induced male reproductive toxicity. In addition, the study also showed that the inhibitory effect of ROF on PDE4 can activate the cAMP/Nrf2/HO-1 and NQO-1 signaling pathways, which play an important role in alleviating CIS-induced oxidative damage and inflammatory response as well as testicular damage. In addition, ROF alone or in combination with CIS can induce apoptosis in prostate cancer PC3 cells by increasing caspase-3, Bax protein and gene expression and Bax/Bcl-2 ratio, and decreasing Bcl-2 protein and gene expression. This effect can be attributed to the stimulation of Nrf2/HO-1 expression and the downregulation of NF-κB p65 gene expression. Therefore, this study provides a new and promising strategy to enhance the anticancer effect of CIS and further reduce its testicular toxicity by combining it with ROF. Further research is needed to evaluate the potential clinical application value of this combination regimen 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.)