A multi-target antisense approach against PDE4 and PDE7 reduces smoke-induced lung inflammation in mice

Abstract

Background: Recent development in the field of COPD has focused on strategies aimed at reducing the underlying inflammation through selective inhibition of the phosphodiesterase type IV (PDE4) isoform. Although the anti-inflammatory and bronchodilator activity of selective PDE4 inhibitors has been well documented, their low therapeutic ratio and dose-dependent systemic side effects have limited their clinical utility. This study examined the effect of 2'-deoxy-2'-Fluoro-beta-D-Arabinonucleic Acid (FANA)-containing antisense oligonucleotides (AON) targeting the mRNA for the PDE4B/4D and 7A subtypes on lung inflammatory markers, both in vitro and in vivo.

Methods: Normal human bronchial epithelial (NHBE) cells were transfected with FANA AON against PDE4B/4D and 7A alone or in combination. mRNA levels for target PDE subtypes, as well as secretion of pro-inflammatory chemokines were then measured following cell stimulation. Mice were treated with combined PDE4B/4D and 7A AON via endo-tracheal delivery, or with roflumilast via oral delivery, and exposed to cigarette smoke for one week. Target mRNA inhibition, as well as influx of inflammatory cells and mediators were measured in lung lavages. A two-week smoke exposure protocol was also used to test the longer term potency of PDE4B/4D and 7A AONs.

Results: In NHBE cells, PDE4B/4D and 7A AONs dose-dependently and specifically inhibited expression of their respective target mRNA. When used in combination, PDE4B/4D and 7A AONs significantly abrogated the cytokine-induced secretion of IL-8 and MCP-1 to near baseline levels. In mice treated with combined PDE4B/4D and 7A AONs and exposed to cigarette smoke, significant protection against the smoke-induced recruitment of neutrophils and production of KC and pro-MMP-9 was obtained, which was correlated with inhibition of target mRNA in cells from lung lavages. In this model, PDE AONs exerted more potent and broader anti-inflammatory effects against smoke-induced lung inflammation than roflumilast. Moreover, the protective effect of PDE4B/4D and 7A AON was maintained when a once-weekly treatment schedule was used.

Conclusion: These results indicate that inhaled AON against PDE4B/4D and 7A have unique effects on biomarkers that are believed to be important in the pathophysiology of COPD, which supports further development as a potential therapy in this disease.

Figure 1

Effect of 4B/4D and 7A antisense oligonucleotides (AON) on target mRNA expression in NHBE cells. NHBE cells were transfected for 24 h with AON 4B/4D, with AON 7A, or with control sequences (CTRL) at 67, 134 or 267 nM, or were not transfected (NT). 4 h before the end of the transfection period, fresh media containing TNF-α, IL-1β and IFN-γ was added. mRNA levels for (A) PDE4B, (B) PDE4D and (C) PDE7A were quantified by real-time PCR analysis and normalized to a reference gene (Ppib). Results are expressed as percentage of PDE mRNA levels in non-transfected cells (± SEM). *: p < 0.05; **: p < 0.01, ***: p < 0.001, t-test relative to CTRL (n = 3 to 9 replicates).

Figure 2

Inhibition of inflammatory mediators by combined 4B/4D and 7A AONs in NHBE cells. NHBE cells were transfected for 24 h with combined 4B/4D and 7A AON or with control sequences (CTRL) at 134 or 267 nM each, or were not transfected (NT). 4 h before the end of the transfection period, fresh media containing TNF-α, IL-1β and IFN-γ was added. Non-stimulated, non-transfected cells are shown as NS-NT. IL-8 (A) and MCP-1 (B) proteins were measured in cell culture supernatants by ELISA. Results are expressed as percentage of chemokine levels in non-transfected cells (± SEM). **: p < 0.01; ***: p < 0.001; t-test versus NS-NT or versus CTRL, as indicated (n = 6 replicates).

Figure 3

Study protocols for cigarette smoke exposure in mice. A) For the one-week smoke model protocol, mice were treated by endo-tracheal delivery of combined 4B/4D + 7A AON on days 1, 3, 6 and 8. On days 6 to 9, other groups of mice were treated daily with oral roflumilast. On days 6 to 9 (3 h after AON or roflumilast treatment), mice were nose-only exposed to the smoke of two 2R4f reference cigarettes per day. Bronchoalveolar lavages (BAL) were performed on Day 10. B) For the two-week smoke model protocol, mice were treated by endo-tracheal delivery of combined 4B/4D + 7A AON daily from Day 1 to Day 5, then once a week on Day 8 and 15. On days 8 to 12 and 15 to 18, mice were nose-only exposed to the smoke of two 2R4f reference cigarettes per day. BAL were performed on Day 19.

Figure 4

Efficacy of treatment with combined 4B/4D and 7A AONs against cigarette smoke-induced lung inflammation. Mice were treated with endo-tracheal vehicle only (VEH), with combined 4B/4D and 7A AON (0.008, 0.04 or 0.2 mg/kg/day) or with control sequences (CTRL, 0.2 mg/kg/day) and exposed to cigarette smoke for one week, as per protocol design described in Figure 3A. Neutrophil counts (A), KC (B) and pro-MMP-9 levels (C) were measured in BAL. Results are expressed as percentage of neutrophils, KC or pro-MMP-9 levels in vehicle-treated and smoke-exposed animals (± SEM). *: p < 0.05; ***: p < 0.001; t-test versus vehicle-treated without smoke exposure, or versus CTRL, as indicated (compilation of 4 independent experiments; n = 10–20 mice per group for neutrophils, 14–15 per group for KC, and 9–10 per group for pro-MMP-9).

Figure 5

Target mRNA expression in BAL cells following treatment with 4B/4D and 7A AON and smoke exposure. Mice were treated with combined 4B/4D and 7A AON (0.008, 0.04 or 0.2 mg/kg/day) or with control sequences (CTRL, 0.2 mg/kg/day) and exposed to cigarette smoke for one week, as per protocol design described in Figure 3A. PDE4B (A), PDE4D (B) and PDE7A (C) mRNA expression were quantified in BAL cells lysates using the Quantigene^® assay, and normalized to the expression of GAPDH used as a reference gene. Results are expressed as percentage of PDE mRNA levels in vehicle-treated and smoke-exposed animals (± SEM). *: p < 0.05; **: p < 0.01; ***: p < 0.001; t-test versus CTRL (compilation of 4 independent experiments, n = 10 to 20 mice per group).

Figure 6

Effect of treatment with roflumilast on cigarette smoke-induced lung inflammation. Mice were treated with oral vehicle only or with oral roflumilast (5 mg/kg/day) and exposed to cigarette smoke for one week, as per protocol design described in Figure 3A. Neutrophil counts (A), KC (B) and pro-MMP-9 levels (C) were measured in BAL. Results are expressed as percentage of neutrophils, KC or pro-MMP-9 levels in vehicle-treated and smoke-exposed animals (± SEM). ***: p < 0.001; t-test versus vehicle-treated without smoke exposure (compilation of 4 independent experiments, n = 12–21 mice per group for neutrophils and KC, and 3–6 per group for pro-MMP-9).

Figure 7

Sustained effect of 4B/4D and 7A AONs following once a week treatment and two weeks of smoke exposure. Mice were treated with endo-tracheal vehicle only (VEH), with combined 4B/4D and 7A AON (0.05, 0.1 or 0.2 mg/kg/day) or with control sequences (CTRL, 0.2 mg/kg/day) daily for one week, then once a week during the 2-week smoke exposure period, as per protocol design described in Figure 3B. Neutrophil counts (A), KC (B) and pro-MMP-9 levels (C) were measured in BAL. Results are expressed as percentage of neutrophils, KC or pro-MMP-9 levels in vehicle-treated and smoke-exposed animals (± SEM). *: p < 0.05; **: p < 0.01; ***: p < 0.001; t-test versus vehicle-treated without smoke exposure, versus vehicle-treated or versus CTRL, as indicated (n = 4 to 8 mice per group).