PDE4

In vitro, cigarette smoke extract (CSE)-induced epithelial-mesenchymal transition (EMT) in WD-HBEC was partially inhibited by roflumilast N-oxide at a concentration of 2 nM. After CSE, 45% of the reduced E-cadherin transcript expression is restored by roflumilast N-oxide (2 nM). The expression of type I collagen is eliminated by roflumilast N-oxide (2 nM). There seems to be protection for the epithelial cell phenotype when roflumilast N-oxide (2 nM) was added to the cells. Additionally, β-catenin nuclear translocation is partially attenuated by preincubation with roflumilast N-oxide (2 nM) [2].

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Neutrophil chemotaxis is involved in the lung inflammatory process in conditions such as chronic obstructive pulmonary disease (COPD). Neutrophil elastase (NE), one of the main proteases produced by neutrophils, has an important role in the inflammatory process via the release of chemokines from airway epithelial cells. It was recently shown that Roflumilast N-oxide has therapeutic potential in COPD. The aim of the present study was to investigate roflumilast N-oxide's effect on NE-induced chemokine production and signaling pathways in A549 epithelial cells. A549 cells were incubated with NE for 30min, washed with PBS and then cultured for 2h (for measurement of mRNA expression) and 24h (for chemokine release) or for 5 to 30min (for protein phosphorylation assays). Prior to the addition of NE, cells were also pre-incubated with prostaglandin E2 (PGE2), alone and in combination with roflumilast N-oxide. Addition of NE was associated with elevated chemokine production by A549 cells and induction of the p38α pathway. In contrast when combined with PGE2, the roflumilast N-oxide had an additive effect on the inhibition of NE-induced chemokine release and p38α and other kinases activation. In conclusion, we demonstrated that NE is able to increase the release of chemokines from epithelial cells via the activation of p38α MAP-kinase and that roflumilast N-oxide when combined with PGE2 lowers NE-induced kinase activation and chemokine production. [1]

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Effects of a combination of Roflumilast N-oxide/RNO and PGE2 on NE-induced chemokine release by A549 epithelial cells [1]
Roflumilast N-oxide alone had no effect on NE-induced chemokine release (data not shown). In contrast, the combination of RNO with 10 nM PGE2 decreases IL-8/CXCL8, MCP1/CCL2 and Gro-α/CXCL1 release. The incubation of epithelial cells with PGE2 alone is able to decrease of NE-induced IL-8/CXCL8, MCP1/CCL2 and Gro-α/CXCL1 release (Fig. 4A, B and C), however the combination of RNO with 10 nM PGE2, further reduced the release of MCP1/CCL2 and Gro-α/CXCL1 (Fig. 4B and C) with respect to PGE2.

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Activation of the p38α kinase pathway by NE is less intense after treatment with Roflumilast N-oxide/RNO [1]
The treatment of epithelial cells with 10 nM PGE2 (whether alone or in combination with RNO) markedly reduced the NE-induced activation of p38α kinase after 5 min (Fig 6A). However, only the combination of RNO and PGE2 was associated with significantly lower p38α activation (Fig. 6A). This finding was confirmed by the results of a specific ELISA for phosphorylated p38α (Fig. 6B). Whether alone or in combination with RNO, PGE2 did not alter the activation of ERK 1/2 and JNK pathways or the total amount of protein

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Effects of Roflumilast N-oxide/RNO on NE-induced protein phosphorylation in A549 cells [1]
We first used a human phosphoprotein array kit to establish whether or not other signaling pathways were activated by NE 10 nM in A549 cells pre-treated with PGE2. We then analyzed the effects of a combination of RNO and PGE2, relative to PGE2 alone. After the pre-treatment of A549 cells with PGE2, incubation with NE was associated with activation (an increase of 30% compared to the control was considered) of 12 kinases (gray bars; RSK 1/2/3, c-Jun, Akt T308, Hck, Fak, STAT5 a/b, p53 S46, p70 S6, PLC-γ1, Chk2, Pyk2, and PRAS 40 t) of the 45 tested (Fig. 7A). Of the 12 kinases activated by NE in the presence of PGE2, five (blanc bars; Hck, FAK, STAT5 a/b, P70s6 and CHK-2) appeared to be less strongly activated when PGE2 was combined with 1 μM RNO (Fig. 7A and B).

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Background: Cigarette smoking contributes to epithelial-mesenchymal transition (EMT) in COPD small bronchi as part of the lung remodeling process. We recently observed that Roflumilast N-oxide (RNO), the active metabolite of the PDE4 inhibitor roflumilast, prevents cigarette smoke-induced EMT in differentiated human bronchial epithelial cells. Further, statins were shown to protect renal and alveolar epithelial cells from EMT. Objectives: To analyze how RNO and simvastatin (SIM) interact on CSE-induced EMT in well-differentiated human bronchial epithelial cells (WD-HBEC) from small bronchi in vitro. Methods: WD-HBEC were stimulated with CSE (2.5%). The mesenchymal markers vimentin, collagen type I and α-SMA, the epithelial markers E-cadherin and ZO-1, as well as β-catenin were quantified by real time quantitative PCR or Western blotting. Intracellular reactive oxygen species (ROS) were measured using the H2DCF-DA probe. GTP-Rac1 and pAkt were evaluated by Western blotting. Results: The combination of RNO at 2 nM and SIM at 100 nM was (over) additive to reverse CSE-induced EMT. CSE-induced EMT was partially mediated by the generation of ROS and the activation of the PI3K/Akt/β-catenin pathway. Both RNO at 2 nM and SIM at 100 nM partially abrogated this pathway, and its combination almost abolished ROS/ PI3K/Akt/β-catenin signaling and therefore EMT. Conclusions: The PDE4 inhibitor roflumilast N-oxide acts (over)additively with simvastatin to prevent CSE-induced EMT in WD-HBEC in vitro. [2]

Plasma glucagon-like peptide-1 (GLP-1) increased four-fold in db/db mice treated once with 10 mg/kg Roflumilast N-oxide. Roflumilast N-oxide at a dose of 3 mg/kg was found to stop the disease from progressing in db/db mice when given over an extended period of time. When roflumilast-N-oxide was used as a carrier, it preserved islet shape and lowered blood glucose and HbA1c increases by 50% and 50%, respectively. It also doubled fasting serum insulin. Moreover, in primary islets, roflumilast-N-oxide improved forskolin-induced insulin release. Additionally, compared to its parent molecule, rolumilast-N-oxide has stronger hypoglycemia effects [3].

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Single treatment of db/db mice with 10 mg/kg roflumilast or roflumilast-N-oxide enhanced plasma GLP-1 2.5- and fourfold, respectively. Chronic treatment of db/db mice with roflumilast or Roflumilast N-oxide at 3 mg/kg showed prevention of disease progression. Roflumilast-N-oxide abolished the increase in blood glucose, reduced the increment in HbA(1c) by 50% and doubled fasted serum insulin compared with vehicle, concomitant with preservation of pancreatic islet morphology. Furthermore, roflumilast-N-oxide amplified forskolin-induced insulin release in primary islets. Roflumilast-N-oxide showed stronger glucose-lowering effects than its parent compound, consistent with its greater effect on GLP-1 secretion and explainable by pharmacokinetic/pharmacodynamic modelling. Conclusions/interpretation: Our results suggest that roflumilast and Roflumilast N-oxide delay the progression of diabetes in db/db mice through protection of pancreatic islet physiology potentially involving GLP-1 and insulin activities. [3]

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Effect of single administration of roflumilast and Roflumilast N-oxide on plasma GLP-1 [3]
We investigated the effect of a single dose of 10 mg/kg roflumilast or roflumilast-N-oxide on plasma GLP-1 in fasted db/db mice. The PDE4 inhibitor’s effect on plasma GLP-1 was investigated in the presence and absence of a glucose bolus as a physiological initiator of GLP-1 secretion. In the presence of glucose, roflumilast and roflumilast-N-oxide significantly increased plasma GLP-1 by a factor of 2.5 and 4.3 vs vehicle, respectively (Fig. 1). The increase in plasma GLP-1 was observed 10 min after administration of PDE4 inhibitor and glucose, and was reversed 60 min after administration. In the absence of glucose, treatment with roflumilast or roflumilast-N-oxide resulted in minor insignificant increases of plasma GLP-1 (data not shown).

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Effect of chronic administration of roflumilast and Roflumilast N-oxide on body weight and food and water intake [3]
In db/db mice, roflumilast and roflumilast-N-oxide at 3 mg kg–1 day–1 reduced food and water consumption compared with vehicle (Fig. 2a, b). Average food consumption over 4 weeks was reduced by about 17% compared with vehicle after treatment with roflumilast or roflumilast-N-oxide. Water consumption increased during the study period from 6.7 g/day to 15.6 g/day in vehicle-treated animals and declined to 7.6 g/day and 3.8 g/day after 4 weeks of treatment with roflumilast and roflumilast-N-oxide, respectively. The effect on food and water consumption occurred right after the first administration and persisted for the whole study period. Roflumilast and roflumilast-N-oxide showed no effect on body weight (Fig. 2c).

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Effect of chronic administration of roflumilast and Roflumilast N-oxide on blood variables [3]
In vehicle-treated animals, HbA1c almost doubled from 4.4% (24.6 mmol/mol) to 8.2% (66.1 mmol/mol) within 4 weeks. Treatment with 3 mg kg–1 day–1 roflumilast and roflumilast-N-oxide reduced the increase in HbA1c. The effect was visible after 2 weeks of treatment and resulted in a 50% reduction compared with vehicle after 4 weeks of treatment with roflumilast-N-oxide (p < 0.01) (Fig. 3a). Concomitant with HbA1c development, blood glucose increased in vehicle-treated animals (Fig. 3b) by 38% and 51% with respect to glucose AUC−15–60 (Fig. 3c) and fasted glucose (Fig. 3d) at day 28. Roflumilast-N-oxide lowered glucose AUC−15–60 (p < 0.001 vs vehicle) and fasting blood glucose (p < 0.01 vs vehicle) to levels as low as at study start, whereas the effect with roflumilast was only significant for glucose AUC−15–60 (p < 0.01 vs vehicle) (Fig. 3b–d). Fasted serum insulin slightly decreased in vehicle-treated animals from 602 pmol/l at day −1 to 551 pmol/l at day 28. Roflumilast-N-oxide at 3 mg kg–1 day–1 almost doubled fasted serum insulin following 4 weeks of treatment (p < 0.05 vs vehicle). The effect of roflumilast on fasted serum insulin was less pronounced and not significant (Fig. 3e). Glucose-stimulated serum insulin levels did not increase but rather declined 15 min after glucose stimulation in vehicle- and PDE4i-treated animals (data not shown).

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Effect of chronic administration of roflumilast and Roflumilast N-oxide on pancreatic morphology [3]
Histopathological examination of H&E stained slides showed mild to moderate islet atrophy in 11-week-old vehicle-treated db/db mice. The percentage of atrophic islets ranged between 25% and 50%, resulting in an islet atrophy severity grade of 2.6 (Fig. 4). Insulin staining on serial sections demonstrated that islet atrophy was associated with a loss of insulin-producing beta cells (Fig. 5a, b). Treatment with 3 mg/kg roflumilast or roflumilast-N-oxide once daily for 4 weeks preserved pancreatic islet morphology and insulin production in beta cells (Fig. 5c, d), resulting in mild (severity grade 2.0, p > 0.05 vs vehicle) and minimal (severity grade 1.3, p < 0.01 vs vehicle) islet atrophy for roflumilast and roflumilast-N-oxide, respectively (Fig. 4).

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Roflumilast N-oxide amplifies insulin secretion in mouse islets [3]
We examined elevated insulin secretion in response to roflumilast-N-oxide in primary mouse islets (Fig. 7). Forskolin, a broad activator of adenylyl cyclases thereby increasing intracellular cAMP, induced insulin secretion fourfold in mouse islets. The response to 1 μmol/l forskolin was synergistically enhanced with either 10 or 100 nmol/l roflumilast-N-oxide at 10 nmol/l glucose leading to a 5.5- or 7-fold increase in insulin release, respectively. Roflumilast (data not shown) and roflumilast-N-oxide without a co-stimulus were unable to induce insulin secretion in primary mouse islets at various tested glucose conditions (data shown for 100 nmol/l roflumilast-N-oxide at 10 mmol/l glucose).

Treatments [1]
A549 cells were washed and cultured overnight in serum-free F-12 K medium supplemented with antibiotics, l-glutamine and HEPES. The starved cells were incubated with NE for 30 min or vehicle (PBS), washed with PBS and then cultured in serum free F-12 K. After stimulation, cell supernatants were collected at 24 h (for cytokine measurements) and cell pellets were collected after 2 h (for mRNA expression analysis). Alternatively, A549 cells were pre-incubated for 2 h with PGE2 (10 nM) alone or in combination with Roflumilast N-oxide (at 0.1 μM, 0.3 μM and 1 μM), vehicle (DMSO 0.01%) or EGFR inhibitor AG-1478 prior to the addition of NE. All experiments were performed in serum-free medium in triplicate and were repeated at least three times. At the end of the incubation period, culture supernatants were harvested and stored at − 80 °C until further analysis.

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Human phosphoprotein array [1]
Cells were also pre-incubated with PGE2 (10 nM) alone or in combination with Roflumilast N-oxide (1 μM) for 2 h before the addition of NE. Cell lysates (500 μg of total protein per array) were applied to the phosphoprotein array (the Proteome Profiler Human Phosphokinase Array Kit) according to the manufacturer's instructions.

Acute studies in db/db mice [3]
\nAt 7 weeks of age, 16 h fasting mice received a single oral dose of vehicle (4% methocel) or test compound (10 mg/kg roflumilast or Roflumilast N-oxide, maximal pharmacological effective dose without side effects following single administration), and a glucose bolus of 2 g/kg body weight was co-administered as a physiological initiator for GLP-1 secretion. The glucose concentration of 2 g/kg body weight was identified in pre-experiments as being optimal because it did not markedly induce GLP-1 levels on its own but potently triggered GLP-1 release in concert with our PDE4 inhibitors. Plasma GLP-1 was analysed 60 min before, and 10 and 60 min after administration of PDE4 inhibitor and glucose. The effect of roflumilast and Roflumilast N-oxide on plasma GLP-1 was also investigated in the absence of the glucose bolus.\n\nChronic study in db/db mice [3]
\nAt 7 weeks of age, all animals were treated once daily by oral gavage with vehicle (4% methocel), roflumilast or Roflumilast N-oxide. Body weight and food and water intake were monitored daily. Animals destined for pharmacodynamic analysis (PD animals) were treated with roflumilast (0.3, 1 and 3 mg kg–1 day–1) or Roflumilast N-oxide (3 mg kg–1 day–1) for 28 days, with 3 mg/kg being the maximal pharmacological effective dose without side effects following chronic administration. HbA1c was determined 6 days and 1 day before, and 14 and 28 days after treatment start. At days 1 and 28, an OGTT was performed for analysis of blood glucose and serum insulin. For OGTT, animals received a glucose bolus of 1 g/kg body weight after fasting for 4 h. In contrast to 16 h fasting in the acute study, the shorter 4 h fasting period results from method optimisation experiments we performed for ethical reasons and provides comparable results to those with 16 h fasting. Blood was collected 15 min before and 15 and 60 min after the glucose challenge. The glucose AUC was determined from time −15 to 60 min. Additional blood samples were taken at days 20 and 28 after treatment start for drug exposure analysis. Animals were killed by cervical dislocation on day 28 and the pancreas was removed for histopathological examination. For pharmacokinetic (PK) analysis, we included satellite animals (PK animals) which were treated with 3 mg kg–1 day–1 roflumilast or Roflumilast N-oxide for 34 days. At days 1, 14, 20, 34 and 35, blood samples were taken for drug exposure analysis.\n\nPKPD analysis in chronic db/db mouse study [3]
\nDrug exposure was analysed in blood samples from PD and PK animals taken at different time points, with each animal providing two and eight samples, respectively (PD animals: −0.5/2 h, day 20; 30 h, day 28. PK animals: 0.5/1/2/4/8 h, day 1; 0.5/1/4 h, day 14; 0.5/2 h, day 20; −0.5/0.5/1/2/4/8 h, day 34; 30 h, day 35). Samples with values greater than the lower limit of quantification (LLOQ) were used for analysis (HPLC-MS/MS, LLOQ = 0.5 μg/l) after Roflumilast N-oxide dosing, corresponding to 51 and 60 concentration values being available for parent and metabolite, respectively. After roflumilast dosing, 89 concentration values were available for both parent and metabolite, and values below LLOQ were set to half LLOQ (0.25 μg/l) to reduce model bias. Four PK models were developed, that is for both roflumilast and its metabolite after both roflumilast and Roflumilast N-oxide dosing, respectively. All variables were estimated using the nonlinear mixed-effects modelling (NONMEM) technique (version 7, ICON Development Solutions, Ellicott City, MD, USA) with first-order conditional estimation with interaction. From the obtained individual apparent clearance estimates, individual steady state AUC was calculated for each PD animal according to the following equation: AUC (μg h l−1) = dose (μg/kg)/clearance (l h−1 kg−1). AUC estimates were used to determine total PDE4 inhibition (tPDE4i) of the respective compounds for all PD animals. The tPDE4i relates the average free concentration of a compound in plasma to its in vitro IC50 of PDE4 inhibition, and is an exposure surrogate allowing for the consideration of parallel contribution of parent and metabolite to the overall effect. [3]
\nwith rof and rofNO corresponding to roflumilast and Roflumilast N-oxide, fu corresponding to the unbound fraction in mouse plasma in vitro, IC50 corresponding to the compound concentration resulting in 50% PDE4 inhibition in vitro and τ corresponding to the dosing interval. To consider the presence of both roflumilast and roflumilast-N-oxide in the circulation, tPDE4i values calculated separately for each compound were added. The combined individual tPDE4i values were used as a measure of exposure in the subsequent PKPD model. HbA1c levels on days −1, 14 and 28 from vehicle, roflumilast and roflumilast-N-oxide groups were used as PD readout, and were related to individual tPDE4i values using the following equation:

[1]. Roflumilast n-oxide associated with PGE2 prevents the neutrophil elastase-induced production of chemokines by epithelial cells. Int Immunopharmacol. 2016 Jan;30:1-8.

[2]. Simvastatin Increases the Ability of Roflumilast N-oxide to Inhibit Cigarette Smoke-Induced Epithelial to Mesenchymal Transition in Well-differentiated Human Bronchial Epithelial Cells in vitro. COPD. 2015 Jun;12(3):320-31.

[3]. The glucose-lowering effects of the PDE4 inhibitors roflumilast and roflumilast-N-oxide in db/db mice. Diabetologia. 2012 Oct;55(10):2779-2788.

We found that NE can increase the release of epithelial cell chemokines by activating p38α MAP kinase. In addition, we found that EGF receptor inhibitors can inhibit the production of NE-induced IL-8/CXCL8 and Gro-α/CXCL1, but have no effect on the production of MCP-1/CCL2. Conversely, treatment of cells with RNO and PGE2 reduced the release of IL-8/CXCL8, MCP-1/CCL2 and Gro-α/CXCL1 and reduced the activation of multiple kinases. Our results confirm the anti-inflammatory effect of RNO in this in vitro elastase model and help to explain the mechanism of action of this compound. [1] Roflumilast N-oxide and simvastatin may inhibit the increase of CSE-induced GTP binding to Rac1 through a complementary mechanism, thereby producing an additive effect. Statins inhibit GDP→GTP exchange by limiting geraniol geraniolization, thereby inhibiting Rac1 membrane binding to PDE4 inhibitors. Given the crucial role of GTP-Rac1 in NOX1/NOX2 activity, the additive effect of roflumilast N-oxide and simvastatin in inhibiting CSE-induced ROS may reflect their inhibitory effect on GTP-Rac1. As previous studies have shown, ROS scavenging can protect WD-HBECs from CSE-induced EMT damage (Reference 4). Therefore, the additive effect of roflumilast N-oxide and simvastatin in preventing CSE-induced EMT may be attributed to their inhibitory effect on CSE-induced ROS in WD-HBECs. The fact that simvastatin and roflumilast N-oxide can inhibit CSE-induced PI3K/Akt pathway activation and the resulting increase in nuclear β-catenin in WD-HBECs is a novel finding. However, a plausible explanation might be their ability to attenuate CSE-induced ROS in WD-HBECs. In summary, this study demonstrates that the PDE4 inhibitor roflumilast N-oxide and the HMG-CoA reductase inhibitor simvastatin have an additive effect in reducing tobacco smoke-induced differentiation of well-differentiated human bronchial epithelial cells (EMT). PDE4 inhibitors and simvastatin may act on different pathways in CSE-induced EMT, manifested as inhibition of GTP-Rac1, ROS, PI3K/Akt and nuclear β-catenin. [2] To the best of our knowledge, we have demonstrated the effects of PDE4 inhibitors in a type 2 diabetes animal model for the first time, with a detailed assessment of disease progression and an analysis of their effects on key glucose metabolism hormones GLP-1 and insulin. Roflumilast is a selective PDE4 inhibitor that has previously been approved for the treatment of COPD. A recent human study of roflumilast explored its efficacy in type 2 diabetes (EFM Wouters, Maastricht University Medical Center, Netherlands, unpublished results). In humans, roflumilast is metabolized to its active metabolite, roflumilast N-oxide, at levels nearly 10 times higher than the parent compound, and is the main component responsible for roflumilast's pharmacological effects. Since the metabolism of roflumilast in db/db mice is unclear, we established a roflumilast N-oxide treatment group to ensure effective plasma concentrations. Consistent with previously reported effects of rolipram on increasing GLP-1 levels in non-diabetic rats, we now confirmed this result in a type 2 diabetes model. In diabetic db/db mice, a single oral dose of 10 mg/kg roflumilast or its metabolite, in the presence of glucose, increased plasma GLP-1 levels by 2.5-fold and 4.3-fold, respectively, compared to the control group. Since this study also showed that rolipram directly stimulates cAMP-mediated GLP-1 release in GLUTag cells, we conclude that the observed roflumilast-induced GLP-1 elevation is due to cAMP-mediated GLP-1 release in the intestinal L cells of db/db mice. In the absence of glucose, roflumilast and roflumilast N-oxide had no significant effect on plasma GLP-1, indicating that PDE4 inhibitors enhance GLP-1 release only after food intake (a physiological initiator of GLP-1 secretion). In contrast, therapeutic GLP-1 analogs increased GLP-1 levels regardless of food intake. Long-term treatment with roflumilast or roflumilast N-oxide significantly improved the diabetic status of db/db mice. Roflumilast N-oxide at a dose of 3 mg kg–1 day–1 almost completely inhibited the increase in area under the glucose curve (AUC) and fasting blood glucose during the study period, and reduced the increase in glycated hemoglobin (HbA1c) by approximately 50% compared to the control group. Furthermore, after 4 weeks of treatment, fasting serum insulin levels nearly doubled, while islet morphology improved and β-cell insulin secretion function was maintained. Improvement in diabetic status was also reflected in reduced water intake and food intake (albeit to a lesser extent). Water intake decreased to 3.8 g/day, the same level observed in healthy mice (data not shown), indicating that the kidneys were no longer overloaded in glucose reabsorption, a stark contrast to the control group's water intake of up to 15.6 g/day at the end of the study. Although roflumilast-N-oxide reduced food intake, it had no effect on weight gain. However, this difference may be a specific effect of db/db mice, as previous studies have shown that PDE4 inhibitors can reduce weight in diet-induced obese mice and diabetic COPD patients. This suggests that PDE4 inhibitors may be more advantageous than currently used hypoglycemic agents such as insulin, sulfonylureas, or thiazolidinones, which typically lead to weight gain. Long-term use of roflumilast-N-oxide produced a stronger hypoglycemic effect compared to the parent drug roflumilast, manifested by significant reductions in blood glucose and glycated hemoglobin (HbA1c) levels and better pancreatic islet cell protection. The two compounds had similar effects on food intake; however, this may be due to the limited sensitivity of the method. The enhanced hypoglycemic effect following long-term use of roflumilast N-oxide is consistent with its stronger GLP-1 elevation effect following acute use. This observation, along with a large body of published literature on the hypoglycemic effects of GLP-1, supports the view that roflumilast-mediated GLP-1 elevation is associated with the prevention of diabetes progression in db/db mice. In particular, treatment of db/db mice with GLP-1 mimics yielded results similar to our findings, showing reduced food and water consumption and protection of the islets of Langerhans. Another impressive observation supports our interpretation that PDE4 inhibitors may improve diabetes by enhancing physiological intestinal GLP-1 release. Comparison of the pharmacodynamic profiles of GLP-1 mimics and PDE4 inhibitors in humans showed that both treatments reduced body weight and caused gastrointestinal side effects such as delayed gastric emptying, transient nausea, and diarrhea.
The reason why roflumilast N-oxide has a stronger hypoglycemic effect compared with roflumilast is that the pharmacokinetic model showed that the value of the combined exposure replacement indicator tPDE4i after treatment with roflumilast N-oxide was 1.6 times higher than that of the parent drug roflumilast. In addition, the model results showed that, based on the estimated EMAX value of 1, the PDE4 inhibitor could maintain HbA1c at the initial level in db/db mice. Since the estimated PDE50 value of 27.1 in db/db mice is about 27 times higher than the tPDE4i value of 1.03 in humans at an effective dose of 500 μg[30], it seems that db/db mice require a higher drug exposure than humans to achieve the therapeutic effect. This should be taken into account when translating mouse results to humans. [3]

C17H14CL2F2N2O4

419.2069

418.029

C, 48.71; H, 3.37; Cl, 16.91; F, 9.06; N, 6.68; O, 15.27

292135-78-5

Roflumilast;162401-32-3;Roflumilast Impurity E;1391052-76-8

9940999

White to off-white solid powder

1.6±0.1 g/cm3

519.7±50.0 °C at 760 mmHg

181 °C

268.1±30.1 °C

0.0±1.4 mmHg at 25°C

1.616

1.43

1

7

6

27

645

0

C1CC1COC2=C(C=CC(=C2)C(=O)N=C3C(=CN(C=C3Cl)O)Cl)OC(F)F

KHXXMSARUQULRI-UHFFFAOYSA-N

InChI=1S/C17H14Cl2F2N2O4/c18-11-6-23(25)7-12(19)15(11)22-16(24)10-3-4-13(27-17(20)21)14(5-10)26-8-9-1-2-9/h3-7,9,17,25H,1-2,8H2

3-(cyclopropylmethoxy)-N-(3,5-dichloro-1-hydroxypyridin-4-ylidene)-4-(difluoromethoxy)benzamide

roflumilast N-oxide; 292135-78-5; Benzamide, 3-(cyclopropylmethoxy)-N-(3,5-dichloro-1-oxido-4-pyridinyl)-4-(difluoromethoxy)-; F08MQ6CZCS; DTXSID80433059; ROFLUMILAST METABOLITE M07; BYK22890; 3-(Cyclopropylmethoxy)-N-(3,5-dichloro-1-oxido-4-pyridinyl)-4-(difluoromethoxy)benzamide;

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)

DMSO : ~250 mg/mL (~596.36 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.96 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 20.8 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 2: ≥ 2.08 mg/mL (4.96 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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