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].

---

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]

---

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.

---

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

---

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).

---

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].

---

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]

---

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).

---

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).

---

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).

---

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).

---

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.

---

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]
At 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. Chronic study in db/db mice [3]
At 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. PKPD analysis in chronic db/db mouse study [3]
Drug 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]
with 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 is able to increase the release of chemokines from epithelial cells via the activation of p38α MAP-kinase. Moreover, we showed that an EGF receptor inhibitor is able to inhibit NE-induced production of IL-8/CXCL8 and Gro-α/CXCL1 but not of MCP-1/CCL2. In contrast, treatment of the cells with a combination of RNO and PGE2 was associated with lower IL-8/CXCL8, MCP-1/CCl2 and Gro-α/CXCL1 release and a lower degree of activation of several kinases. Our results confirm RNO's anti-inflammatory effect in this in vitro elastolytic model and help to explain the compound's mechanism of action.[1]

---

Roflumilast N-oxide and simvastatin may prevent the CSE-induced increase of GTP-bound Rac1 by complementary mechanisms resulting in an additive interaction. While the statin limits geranylgeranylation hence, Rac1 membrane docking the PDE4 inhibitor would curb the GDP→GTP exchange. Given the critical role of GTP-Rac1 for NOX1/NOX2 activity the additive effects of roflumilast N-oxide and simvastatin to suppress CSE-induced ROS may reflect their inhibition of GTP-Rac1. As previously shown removal of ROS protects from CSE-induced EMT in WD-HBEC (Citation4). Therefore, that roflumilast N-oxide and simvastatin were additive to prevent CSE-induced EMT could be attributed to their suppression of CSE-induced ROS in WD-HBEC.

That simvastatin and Roflumilast N-oxide prevented CSE-induced activation of the PI3K/Akt pathway and the ensuing increase in nuclear β-catenin in WD-HBEC is a novel finding. One plausible explanation however, may be their ability to attenuate CSE-induced ROS in WD-HBEC.

In conclusion the current work shows additive effects of the PDE4 inhibitor Roflumilast N-oxide and the HMG-CoA reductase inhibitor simvastatin to mitigate EMT secondary to tobacco smoke in well-differentiated human bronchial epithelial cells. The PDE4 inhibitor and the statin may act on different pathways involved in CSE-induced EMT reflected by inhibition of GTP-Rac1, ROS, PI3K/Akt and nuclear β-catenin. [2]

---

To our knowledge, we have shown for the first time the effect of PDE4 inhibition in an animal model of type 2 diabetes evaluating disease progression in detail and analysing the effects on GLP-1 and insulin being central hormones in glucose metabolism. Roflumilast was used as a selective PDE4 inhibitor, previously approved as a drug for the treatment of COPD. A recent human study with roflumilast addressed the question of efficacy in type 2 diabetes (E.F.M Wouters, Maastricht University Medical Center, Maastricht, the Netherlands, unpublished results). In humans, roflumilast is metabolised to the active metabolite Roflumilast N-oxide, which exhibits a nearly 10-fold higher exposure compared with its parent compound and acts as the principal contributor to the roflumilast drug effect. As roflumilast metabolism in db/db mice was unknown, we included one treatment arm with roflumilast-N-oxide to assure efficacious plasma concentrations.

In accordance with the previously reported GLP-1 elevating effect of rolipram in non-diabetic rats, we now confirm the result in a type 2 diabetes model. In diabetic db/db mice, single oral administration of 10 mg/kg roflumilast or its metabolite increased plasma GLP-1 vs vehicle in the presence of glucose by a factor of 2.5 and 4.3, respectively. As this report showed in addition that rolipram is able to directly stimulate the cAMP-mediated GLP-1 release in GLUTag cells, we concluded that the observed increase of GLP-1 in response to roflumilast results from cAMP-mediated GLP-1 release in intestinal L-cells of the db/db mice. In the absence of glucose, roflumilast and Roflumilast N-oxide had no significant effect on plasma GLP-1 suggesting that PDE4 inhibition amplifies GLP-1 release only following food intake, which is the physiological initiator of GLP-1 secretion. In contrast, therapeutic GLP-1 mimetics elevate GLP-1 levels independent of food intake.

Chronic treatment of db/db mice with roflumilast or Roflumilast N-oxide clearly ameliorated the diabetic status of the animals. Roflumilast-N-oxide at 3 mg kg–1 day–1 almost abolished the increase in glucose AUC and fasting glucose over the study time and reduced the increment in HbA1c by about 50% vs vehicle. In addition, fasted serum insulin levels almost doubled following 4 weeks of treatment concomitant with amelioration of pancreatic islet morphology and preservation of insulin production in beta cells. The improved diabetic status was also reflected by a reduction in water and, although less pronounced, food consumption relative to control. Water consumption decreased to 3.8 g/day, a level we observed in healthy mice (data not shown), suggesting that the renal glucose reabsorption capacity was no longer overloaded in contrast to vehicle-treated mice showing a high water intake of 15.6 g/day at the end of the study. Despite its food-reducing effect, roflumilast-N-oxide had no effect on body weight development. However, this discrepancy might be a db/db mouse-specific effect as PDE4 inhibitors have been shown to reduce body weight in diet-induced obesity mice and diabetic COPD patients. This indicates that PDE4 inhibitors might be advantageous over current glucose-lowering drugs such as insulin, sulfonylureas or thiazolidinones, for which body weight gain is often an adverse event.

Compared with its parent roflumilast, chronic Roflumilast N-oxide treatment resulted in stronger glucose-lowering effects as demonstrated by a greater reduction of blood glucose and HbA1c and superior islet preservation. The effect on food intake was similar for both compounds; however, this may be attributed to the limited sensitivity of the method. The stronger glucose-lowering effect of roflumilast-N-oxide following chronic treatment is consistent with its greater GLP-1-increasing effect following acute treatment. This observation and extensive published literature regarding the glucose-lowering role of GLP-1 support the view that roflumilast-mediated GLP-1 elevation and the prevention of diabetes progression in our db/db mice are linked. In particular, treatment of db/db mice with GLP-1 mimetics shows parallels to our study results with respect to reduced food and water consumption and preservation of pancreatic islets. Another observation was striking to us, supporting our interpretation that PDE4 inhibitors ameliorate diabetes likely via enhancement of physiological intestinal GLP-1 release. Comparing drug reaction profiles of GLP-1 mimetics and PDE4 inhibitors in humans shows that both treatments can reduce body weight and cause gastrointestinal side effects such as delayed gastric emptying, transient nausea and diarrhoea.

A reason for the stronger glucose-lowering effect of Roflumilast N-oxide compared with roflumilast is given by PK modelling as shown by a 1.6-fold higher value of the combined exposure surrogate tPDE4i after roflumilast-N-oxide treatment compared with treatment with the parent roflumilast. Furthermore, the modelling results suggest that PDE4 inhibitors carry the potential to keep HbA1c at starting levels in db/db mice based on the estimated EMAX of 1. As the estimated PDE50 of 27.1 in db/db mice is approximately 27-fold higher than the tPDE4i of 1.03 in humans at the effective dose of 500 μg [30], db/db mice seem to require higher exposures for efficacy than humans. This should be considered in the translation of results from mice 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.

---

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.

---

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