Background
Chronic obstructive pulmonary disease (COPD) is a complex syndrome characterized by chronic bronchitis, persistent cellular inflammation and progressive deterioration of airways and emphysema [
1‐
3], for which cigarette smoking is by far the most important risk factor [
4]. COPD is one of the leading causes of death and morbidity worldwide [
5]. To date, no therapies have been shown to reduce mortality or prevent disease progression.
Although the composition of the lung cellular infiltrate varies among COPD patients, it is mainly constituted by neutrophils, macrophages and CD8
+ T cells [
6]. The neutrophilic arm of airway inflammation is believed to be at the forefront of the lung pathogenesis in COPD patients [
1,
7‐
9]. In the airways, neutrophils can release a number of mediators including oxygen radicals, elastases and metalloproteases (MMP) that contribute to self-perpetuation of inflammation and promote matrix breakdown, leading to alveolar destruction and emphysema [
10‐
12]. Patients with COPD have an increased number of neutrophils in broncho-alveolar lavages (BAL), sputum, airways and lung parenchyma [
8,
9], which directly correlates with disease severity [
13]. Their recruitment and accumulation in the airways is driven by chemokines such as interleukin-8 (IL-8), the levels of which have been found to be increased in sputum, alveolar macrophages and bronchial epithelium obtained from COPD patients [
14‐
16].
In airways, elevation of intracellular cAMP has been associated with the general suppression of activity of inflammatory cells and relaxation of airway and vascular smooth muscle [
17]. Levels of intracellular cAMP are determined by the enzymatic balance between synthesis by adenylate cyclase and hydrolysis by phosphodiesterases (PDE). The PDEs represent a large family of isozymes [
18], of which PDE4B and PDE4D isotypes are predominantly expressed in a variety of inflammatory and structural lung cells [
17], and have been shown to modulate the inflammatory response [
19,
20]. Small molecule PDE4 inhibitors with broad spectrum anti-inflammatory effects have been shown to reduce inflammatory cell recruitment and improve lung function in animal models of COPD [
21‐
23]. Orally active PDE4 inhibitors such as cilomilast and roflumilast have reached an advanced clinical stage [
24‐
26]. However one major hurdle in their development has been overcoming the dose-limiting systemic side effects, of which headaches, nausea and vomiting are the most common manifestations [
27]. Moreover, arteritis and vasculitis in the gastrointestinal tract and mesenteric blood vessels of rodents [
28] and cardiac tissue of primates [
29] have also raised a concern about their safety profile. Although delivery of PDE4 inhibitors via inhalation could represent an alternative approach [
22,
30], the efficacy and safety of inhaled small molecules PDE4 inhibitors remains to be demonstrated [
31,
32]. Consequently, improving the therapeutic ratio of PDE4 inhibitors still represents an important challenge.
One strategy to overcome these limitations is to develop more selective PDE4 inhibitors. For example, as systemic PDE4D inhibition appears to be responsible for the emetic effect [
33], PDE4B-specific inhibitors might provide better therapeutic ratios. However, as PDE4D also has a predominant role in the activation of T cells [
34], PDE4B-specific inhibitors might display reduced efficacy as compared to inhibitors targeting both isoforms in the lung. Another interesting approach would be to develop inhibitors of other PDE classes to be used in combination with PDE4 inhibitors, with the goal of maintaining or enhancing efficacy while reducing the dose-limiting side effects associated with these drugs [
35]. In this regard, PDE7 might represent a good candidate, as it is widely expressed in lung tissue and inflammatory cells [
36]. Moreover, in vitro studies have shown that the anti-inflammatory activity of the PDE4 inhibitor rolipram was enhanced by a PDE7 inhibitor, although the PDE7 inhibitor alone was without effect [
37].
Antisense oligonucleotides (AON), by their ability to downregulate the expression of specific proteins, represent an innovative therapeutic strategy for lung diseases [
38‐
40]. AONs can inhibit gene expression through formation of duplexes with complementary mRNA, activation of RNAseH and degradation of the duplexes or blockade of the translation machinery [
41‐
43]. The lung is lined with surfactant, which is believed to facilitate AON uptake into cells following inhalation, without the need for specific carriers [
44]. Moreover, inhaled AONs are metabolized mostly in the lung [
45], therefore limiting systemic exposure [
46]. Their clinical utility in lung diseases was illustrated in a recent study where inhaled AONs were used to inhibit the allergen-induced inflammation in asthmatic subjects [
47]. We hypothesized that inhaled AONs against selected PDE subtypes, by downregulating the expression of their targets locally, would reduce inflammation associated with cigarette smoke exposure. We reasoned that a combination of AONs against PDE4 and PDE7 subtypes would mediate improved pharmacological activity as compared to PDE4 only inhibitors. This study therefore examined the
in vitro and
in vivo potency of a combination of phosphorothioate AONs directed against PDE4B/4D and PDE7A subtypes. These AONs incorporate 2'-deoxy-2'-Fluoro-β-D-Arabinonucleic Acid (FANA) modifications, which have been shown to provide increased potency and increased stability as compared to second generation phosphorothioate oligonucleotides [
48,
49].
Discussion
Much interest in the field of COPD has focused on strategies aimed at reducing the underlying inflammation through broad inhibition of the PDE4 isoforms. Orally administered second generation PDE4 inhibitors, such as cilomilast and roflumilast, have undergone extensive investigation and are currently in Phase III clinical development. Although effective, dose-limiting side effects related to the systemic exposure of these drugs have hampered their clinical development. Therefore, there is still a need for more selective and/or more potent PDE inhibitors with improved therapeutic ratios.
Increased interest in the therapeutic use of AONs has been fueled by recent pre-clinical [
38,
44,
53‐
55] and clinical studies [
47] showing their potential benefit in respiratory disorders involving inflammation such as asthma. We have developed FANA-modified antisenses oligonucleotides specifically targeting PDE isotypes 4B, 4D and 7A, as a potential new inhaled drug for the treatment of COPD. In NHBE cells, high levels of PDE4B, 4D and 7A mRNA inhibition were correlated with reduced functional activity, as illustrated by dose-dependent inhibition of IL-8 and MCP-1 secretion. The potency of combined PDE4B/4D and 7A AON on inflammatory markers in vitro is in line with their protective effect in vivo, as evidenced by their ability to reduce the smoke-induced recruitment of neutrophils and secretion of KC and MMP-9 in mice. Inhibition of neutrophil recruitment was also associated with PDE mRNA inhibition in vivo. Interestingly, we observed that the dose required to achieve significant inhibition of the three PDE targets at the mRNA level (0.2 mg/kg/day) was the same as the dose required to block the recruitment of neutrophils, KC and pro-MMP-9. At lower doses where only PDE4B mRNA inhibition was measured, no reduction in neutrophil recruitment was observed (see Figures
4A and
5A). Because PDE4B, 4D and 7A are expressed to different levels in different cell types, these data do not allow us to determine in which cellular subset in the BAL fluid the inhibition of PDE mRNA was the most efficient. The fact that total RNA from the mixed cell populations present in the BAL was measured might reflect this limitation. Nevertheless, these results are consistent with the idea that downregulating different PDE subtypes in different cell populations which together orchestrate the inflammatory response is required to achieve a protective effect against neutrophils in vivo. Moreover, and as previously shown in PDE4B and PDE4D knock out mice [
20], these data support the idea that different PDE isotypes play complementary roles in the control of inflammatory cell recruitment. In the acute smoke exposure protocol used here (1-week model), orally delivered roflumilast at 5 mg/kg/day partially reduced neutrophil recruitment (20% reduction), but statistical significance could not be reached, and had no effect on KC or pro-MMP-9 secretion (Figure
6). Martorana
et al. reported that oral roflumilast at 5 mg/kg inhibited neutrophils by 30% following one acute (20 minutes) exposure to cigarette smoke [
21]. The longer smoke exposure period used here might explain the difference between the studies. Yet, the greater efficacy of the combined AONs PDE4B/4D and PDE7A supports the hypothesis that the anti-inflammatory activity of PDE4 inhibitors might benefit from the addition of a PDE7 inhibitor.
Besides neutrophils, lung macrophages are believed to play an important role in COPD, in part by their ability to release TNF-α, which can drive neutrophil influx and activate MMP-12 release, leading to extracellular matrix degradation [
50]. In both the 1-week and the 2-week smoke models used here, macrophages were significantly increased in lung lavages upon smoke exposure. However, no reduction in macrophage counts were observed in mice treated with PDE4B/4D and 7A AONs, nor with roflumilast (data not shown). Although roflumilast has been shown to reduce macrophage density in lung tissue of mice chronically exposed to smoke (7 months), no effect of roflumilast on macrophages was seen following acute smoke exposure in that study [
21], which is in line with our results. Thus, lack of efficacy on macrophages in the acute smoke exposure protocols used here may reflect the limitations of the acute model, and therefore may not be predictive of the outcome in chronic smoke exposure regimens.
When tested in a two-week smoke exposure protocol, combined PDE4B/4D and 7A AONs (at 0.05 mg/kg/day) were found to significantly reduce neutrophil influx, as well as KC and pro-MMP-9 in lung lavages collected four days after the last AONs treatment (Figure
7). In addition, treatment with combined PDE4B/4D and 7A AONs also resulted in significant inhibition of lymphocyte recruitment (data not shown). Results from these experiments suggest that once a steady state level of AONs is reached in the lung, a once-a-week treatment regimen could be sufficient to keep cellular inflammation to low levels. Although the results from this 2-week smoke exposure protocol are encouraging, more studies are needed to verify whether protection against inflammation is maintained in more chronic smoke exposure models, and whether PDE AON treatment can be beneficial on long term endpoints such as emphysema.
Delivery of AON directly to the lung via inhalation presents key advantages over systemic delivery of small molecule PDE4 inhibitors. Inhalation of AONs achieves appreciable local lung concentrations at the site of action, at lower administered doses. In a 14-day inhalation study in monkeys, PDE-targeting FANA AONs were found to be safe and well tolerated at all dose levels tested (from 0.05 to 2.5 mg/kg/day) [
46]. Moreover, pharmacokinetic studies indicated very low levels of AON in plasma (<1%), and no plasma accumulation was obtained after repeated doses. In humans, there were no detectable levels of AON in plasma following inhalation for four consecutive days of 1.5 mg of TPI ASM8, an AON drug candidate in development for the treatment of asthma [
47].
Competing interests
MF, HD, MEH, KM, SS, RS, PMR and NF are employed by Topigen Pharmaceuticals inc. JG, PA, SM and LP are former employees of Topigen Pharmaceuticals inc. PMR is founder of Topigen, invested 2,200,000.00$ in the company and currently owns approximately 1–2% of company stock. He has many patents received or pending but does not own any royalties. PMR has received a 27,000.00$ grant from Topigen to perform research on RSV.
Authors' contributions
MF and HD participated in the conception, designed the studies, participated in their coordination, analysed the data and drafted the manuscript. MEH, PA, KM, SM and SS carried out in vivo drug treatment and smoke exposure, differential cell counts, mRNA quantification and immunoassays. JG performed in vitro transfections, mRNA quantification and immunoassays. RS, PMR and LP participated in the conception, design and coordination of the studies and critically reviewed the manuscript. NF conceived the studies, participated in their design and coordination, assisted in drafting and critically reviewed the manuscript.