Background
The cyclic nucleotides, cAMP and cGMP are important second messengers known to control many cellular processes, such as inflammation. The cyclic nucleotide signalling system is both complex and interlinked with many other pathways [
1], their signals being tightly controlled by regulating the synthesis and breakdown of these molecules. The only means the cell possesses to degrade the signal mediated by cyclic nucleotides is through the action of a large family of enzymes, the cyclic nucleotide phosphodiesterases [
2].
To date, 11 PDE families have been identified. As these can be derived from multiple genes, many capable of generating a number of isoforms, there currently exists over 50 known PDE enzymes [
3‐
6]. Differences in tissue expression patterns and spatial compartmentalisation within cells resulting in unique functional roles, and controlling mechanisms, is believed to explain the requirement for a large number of enzymes performing a similar process.
Their great diversity, and key role in controlling cyclic nucleotide signalling, makes the PDE family attractive drug targets. In recent years, a number of inhibitors of these enzymes have been brought to the market. Examples of these are the PDE 3 inhibitor, cilostazol, for the treatment of intermittent claudication [
7], and sildenafil, a PDE 5 inhibitor, for the treatment of sexual dysfunction [
8]. The PDE 4 family has also been extensively investigated, as inhibitors of these enzymes are known to be both potent anti-depressants and anti-inflammatory agents [
9,
10]. To date, side effects have prevented PDE4 inhibitors being used commercially, however, a number of groups now appear near to market with specific inhibitors for the treatment of airway diseases [
11‐
13].
Extensive studies using specific inhibitors both
in vitro and
in vivo have demonstrated the regulation of airway smooth muscle by cAMP specific PDE 3 and PDE 4 families [
14‐
16]. Members of both families are also expressed in cells of the immune system and considerable attention has been focussed on the potential of selective inhibitors of PDE 3 and PDE 4, or in combination, for the treatment of asthma and other inflammatory diseases [
17‐
21]. In contrast, despite the presence of the cGMP hydrolysing PDE 5 in airways, the potential anti-inflammatory actions of selective inhibitors has not been fully investigated.
It has been reported that not only do PDE 3 and PDE 4 inhibitors induce relaxation of airways [
22], a combined inhibition of both may produce a more potent anti-inflammatory effect than single inhibition of either alone [
23]. In human alveolar macrophages, endotoxin-induced release of TNF-α is only completely inhibited by a combination of the PDE 3 inhibitor, motapizone, and the PDE 4 inhibitor, rolipram [
24]. In passively sensitised human airways, allergen-induced contraction was inhibited only by the simultaneous inhibition of PDE 3 and PDE 4 and not by single inhibition of either family [
25]. Furthermore, ovalbumin-induced pulmonary eosinophil influx in guinea pigs was attenuated more effectively by a mixed PDE 3/4 inhibitor than by either a selective PDE 3 or PDE 4 inhibitor [
23,
26]. Whether this synergistic relationship is unique to PDE 3 and PDE 4 alone, or if combinations of other PDE inhibitors will produce similar increases in anti-inflammatory activity, remains to be seen. Interestingly, in an
in vivo model of pulmonary resistance, the PDE 3 inhibitor, milrinone, and the PDE 5 inhibitor, zaprinast offer a synergistic effect when used in combination [
27]. To date, however, very little is known regarding the potential interaction between selective inhibitors of PDE 3, PDE 4 and PDE 5.
With PDE inhibitors becoming more commonly used to treat disease, our study sought to determine the effects that combinations of inhibitors may have upon the anti-inflammatory effects of a PDE 4 inhibitor. Therefore, we investigated the abilities of a PDE 3 selective inhibitor (cilostazol), a PDE 4 selective inhibitor (RO 20-1724) and a PDE 5 selective inhibitor (sildenafil), to inhibit inflammation in a murine model of allergic asthma [
28]. The effectiveness of these drugs was evaluated when administered alone and in combination. These results suggest that co-administration of a PDE 3 and/or PDE 5 inhibitor may reduce the anti-inflammatory effectiveness of a PDE 4 inhibitor.
Discussion
The benefits of inhibiting PDEs in disease has been known for many years, however, only recently have drugs emerged that lack the side effects normally associated with these drugs [
11]. Several new PDE 4 inhibitors, possessing little side effects, are nearing launch. In our study, we wished to determine whether there was any synergy in anti-inflammatory function when PDE inhibitors were used in combination. An enhancing effect with one of these combinations, compared to the effects of a PDE 4 inhibitor alone, could suggest this as a possible preferential treatment for inflammatory conditions. A synergistic effect could result in the administration of lower doses of inhibitor, lowering any potential side effects.
A range of PDE inhibitors are about to be launched, or are already in use. Cilostazol, a PDE 3 inhibitor, is being used to treat circulatory problems [
7]. Sildenafil, a PDE 5 inhibitor is used for the treatment of sexual dysfunction and may potentially be used in cardiovascular disease [
30], and there is the imminent release of new PDE 4 inhibitors for inflammatory lung disease [
11,
31]. Due to the success of these PDE inhibitors in the treatment of varied diseases, in future years there exists the potential for a patient to be taking a combination of drugs that will inhibit more than one PDE family.
The results from this study demonstrate that the PDE 4 inhibitor, RO 20-1724, exhibits potent anti-inflammatory actions in our murine model of allergic asthma. Neither the PDE 3 inhibitor, cilostazol, nor the PDE 5 inhibitor, sildenafil, produced any significant anti-inflammatory effect. This conflicts with a recent study that indicated potential benefits of sildenafil in an OVA model [
32]. However, their model used different animals and the administration of the drug was by peritoneal injection, not oral. In addition, the significant anti-inflammatory effects of the PDE 4 inhibitor are reduced upon co-administration with the PDE 3. This is in contrast to many studies that show that a combined PDE3/4 inhibitor has better efficacy over a separate PDE 3 or 4 inhibitor. Our study used a combination of separate PDE 3 and PDE 4 inhibitor and not a dual/mixed compound which posses both PDE 3 and PDE 4 inhibitory potential.
The anti-inflammatory actions of PDE 4 inhibitors are well documented both
in vitro and
in vivo. Selective inhibition of PDE 4 suppresses several eosinophil functions such as superoxide anion generation [
33], adhesion and migration [
34]. Furthermore, PDE 4 inhibitors produce marked anti-inflammatory actions in several animal models of allergic asthma [
21,
22,
35].
Glucocorticoids, such as dexamethasone, are often used as positive control drugs in models of allergic asthma [
36]. Dexamethasone inhibits leukocyte migration into sites of inflammation, predominantly by inhibiting the synthesis of pro-inflammatory cytokines and chemokines [
24]. In this present study, the effects of RO 20-1724 on leukocyte infiltration and pro-inflammatory cytokine levels approached those of dexamethasone, emphasising the potency of PDE 4 inhibitors as anti-inflammatory agents. In our study dexamethasone is able to significantly reduce cell number (total and eosinophil) and inflammatory mediators (IL-5, IL-4 and TNF-α) better or as good as the PDE 4 inhibitor used in the study. Long term, high doses of steroids should be avoided and it may be possible to use PDE4 drugs in combination with lower doses of steroids. We would propose further study into the effects of combining PDE 4 compounds and steroids.
All of the parameters of inflammation that were measured exhibited very similar trends. Thus, the overall pattern of results for cellular infiltration and the levels of the pro-inflammatory cytokines, IL-4, IL-5 and TNF-α, in the BAL fluid were remarkably similar. The Th2-derived cytokines, IL-4 and IL-5 play a central role in initiating and sustaining an asthmatic response by regulating the production of IgE as well as the growth, differentiation and recruitment of inflammatory cells [
37]. Indeed, a positive correlation between lung eosinophilia and levels of IL-4 and IL-5 in BAL fluid has previously been reported, both in humans [
38] as well as animal models of allergic asthma [
39]. Levels of TNF-α are also increased in the airways and BAL fluid of asthmatics relative to healthy subjects [
38,
40], indicating a possible involvement of this pro-inflammatory cytokine in the development of asthma.
Our results are in keeping with previous studies reporting that PDE 4 inhibitors suppress levels of these cytokines. Rolipram inhibits TNF-α production from LPS-stimulated monocytes
in vitro [
41,
42] and from mice
in vivo [
43]. Furthermore, the second-generation PDE 4 inhibitors, Ariflo (SB 207499) and Roflumilast both attenuate TNF-α levels in the BAL fluid from rats and guinea pigs [
31] and inhibit TNF-α, IL-4 and IL-5 production
in vitro [
44].
In some model systems PDE 3 inhibitors have shown the ability to reduce selected markers of inflammation either
in vitro or
in vivo. Cilostazol has been shown to attenuate superoxide anion release from guinea pig alveolar macrophages [
45], while CI-930 was able to decrease eosinophil influx to a similar extent as rolipram in a Brown Norway rat model of asthma [
22]. However, many groups have shown that PDE 3 inhibitors offer no anti-inflammatory potential
in vivo, such as siguazodan [
37], SK&F 94120 [
27] and SK&F 94836 [
13]. Possibly, variation in the models used and inhibition profiles of the these compounds accounts for these observed differences.
Although the anti-inflammatory actions of PDE 5 inhibitors have not been as widely studied as those of PDE 3 or PDE 4 inhibitors, most studies have shown PDE 5 inhibitors to have little or no effect at reversing inflammation, although recent reports may suggest otherwise [
32]. In a recent study, zaprinast failed to inhibit superoxide anion release from guinea pig alveolar macrophages [
16,
45]. Furthermore, rolipram, but not zaprinast, reduced eosinophil recruitment into the lungs in sensitised rats [
22,
46] and guinea pigs [
37].
In this present series of experiments, we have used sildenafil as the inhibitor of cGMP specific PDE 5. Sildenafil is reported to be more selective for PDE 5 than zaprinast and several times more potent, with a low nanomolar IC50 (12). Despite its high selectivity and potency for PDE 5, sildenafil, when used alone, produced no reduction in inflammation in our experiments. These findings are in agreement with the general consensus in that PDE 4 inhibitors are potent anti-inflammatory agents, whereas PDE 3 and PDE 5 inhibitors have little or no effect.
A major aspect of this study was to examine the possible interaction between inhibitors of the PDE 3, PDE 4 or PDE 5 families. More specifically, to examine if a combination of a PDE 3, PDE 4 or PDE 5 inhibitor produced any additive or synergistic benefit as opposed to being used alone. A drawback with the administration of many PDE 4 selective inhibitors in the treatment of asthma is their considerable side effects, such as nausea and stomach acid secretion. Any synergistic or additive effect between a PDE 4 inhibitor and an inhibitor of PDE 3 or PDE 5, may allow lower doses of drug to be administered and thus reduce the severity of any side effects. However, we were unable to demonstrate any enhancement of RO 20-1724-mediated anti-inflammation with either cilostazol, sildenafil or indeed a combination of all three. In contrast, we observed that combining cilostazol with RO 20-1724 resulted in a decrease of the anti-inflammatory actions of the PDE 4 inhibitor. This may be due to a number of reasons such as alterations in PDE4 absorption across the gastrointestinal tract in the presence of PDE3 or PDE5 inhibitor or changes in intracellular signalling pathways.
Authors' contributions
RAC and CAJD carried out the allergic model, writing and preparation of the manuscript and subsequent biochemical analysis. AM carried out the allergic model and assisted with the writing and preparation of the manuscript and subsequent biochemical analysis. MN assisted with the writing and preparation of the manuscript. DG assisted with the allergic model. SFH and SJM participated in the direction of the study as well as writing and preparing the manuscript.