**2.2 Phosphodiesterases as targets for COPD**

436 Chronic Obstructive Pulmonary Disease – Current Concepts and Practice

*usually progressive and associated with an abnormal inflammatory response of the lungs to noxious particles or gases"* (Gold 2009). Symptoms of COPD include chronic cough, excessive sputum production, wheeze, shortness of breath and chest tightness. The 4 stages of COPD, designated as Mild, Moderate, Severe and Very Severe, are defined according to lung function as assessed by spirometry, usually the post-bronchodilator ratio of forced expiratory volume in 1 second (FEV1) to forced vital capacity (FVC). The cellular and molecular mechanisms that contribute to COPD pathogenesis remain incompletely understood yet it is believed that COPD is caused by underlying inflammation characterized by increased presence of neutrophils, macrophages and CD8+ T cells (Gold 2009). Products of neutrophils induce mucus hypersecretion and are implicated both in the generation of mucus metaplasia in chronic bronchitis and the destruction of lung tissue in emphysema. Macrophages are also sources of proteinases and antiproteinases in the lung, oxidative stress and mucus hypersecretion (Ward 2010). Exacerbations play a large role in the disease progression of COPD, and exacerbations become more frequent and more severe

Currently, the only intervention known to influence the loss of lung function is smoking cessation (Gold 2009). Besides treating symptoms and improving quality of life, the treatment focus includes prevention of future exacerbations, reduction of mortality and prevention of disease progression. Treatment for COPD falls into two categories: those medications which relieve symptoms of airflow limitations and those medications which control the underlying inflammation. As such, the current gold standard of treatment for COPD patients involves a step-up paradigm commencing with short-acting bronchodilators (either short-acting 2 agonists or antimuscarinic agents), then adding on long-acting bronchodilators again either long-acting 2 agonists (LABA) or long acting muscarinics, (LAMA) followed by inclusion of inhaled corticosteroids (ICS). Lastly, long term oxygen and possible surgical treatments are final treatment options. Typically, the most common treatment involves ICS/LABA class of drugs, but can also include methylxanthines (bronchodilator) and leukotriene antagonists (anti-inflammatory) (Hurst et al. 2010). The majority of novel treatments for COPD forecasted to launch prior to 2018, are in fact minimally differentiated from current options, with either being improved dosing or

Another dilemma is that although highly effective in asthma, ICS have provided little therapeutic benefit in COPD (Barnes 2006). In patients with severe COPD, histological analysis of their peripheral airways have shown an intense inflammatory response, despite treatment with high doses of ICS, suggesting steroid resistance (Hogg et al. 2004). Combinations of ICS and LABA have been shown to be more effective at reducing COPD exacerbations (Calverley et al. 2007) but have not been shown to statistically decrease mortality (Calverley et al. 2007) (Tashkin et al. 2008). ICS use has been associated with osteoporosis, glaucoma, cataracts and skin thinning (Giembycz &Field 2010) and increased risk of pneumonia in patients with COPD (Ernst et al. 2007). Even with the current and immediate future medications, there are clear unmet needs for more effective antiinflammatories in COPD both for reducing progression of the disease and reducing

as COPD progresses (Hurst et al. 2010).

**2.1 Traditional management of COPD** 

mortality.

combining therapies such as combinations of LABA/LAMA.

PDE4 is a member of the PDE family of enzymes whose function is to selectively catalyze the hydrolysis of cycle adenosine monophosphate (cAMP) and/or cyclic guanosine monophosphate (cGMP) (Bender &Beavo 2006). Second messengers perform intracellular signaling and cAMP is a key member. The level of cAMP can be regulated by its rate of degradation which is controlled by PDEs (Figure 1). As such, the regulation of PDEs is sophisticated and complex. This family currently includes 11 members (PDE1 to PDE11) of which there are multiple isoforms or splice variants. Several different PDEs can be expressed in a single cell type, and the localization of these PDEs within a cell regulates the local concentration of cAMP or cGMP. Besides being regulated through differential genetic expression, PDEs can be biochemically regulated by phosphorylation, binding of Ca2+/calmodulin and various protein-protein interactions (Bender &Beavo 2006). The PDEs with higher affinity for cAMP than cGMP include PDE3, PDE4, PDE7, PDE8 and PDE11 (similar affinities). These multiple isoforms and their differential expression across cell types

Fig. 1. Cartoon of the cAMP pathway, which is presumably activated upon binding of a stimuli to its receptor embedded in the cell membrane. Known components of this pathway include the calcium/calmodulin-activated adenylyl cyclase, the phosphodiesterase (PDE), and cAMP-dependent protein kinase (PKA) with its catalytic and regulatory subunits. Activation of PKA will lead to phosphorylation of cytoplasmic and nuclear targets. In the lung, inhibition of the PDE will lead to an elevation of the intracellular levels of cAMP resulting with a reduction of the bronchoconstriction, mucus secretion, cellular inflammation and in the long term decrease the emphysema/oedema.

A Multi-Targeted Antisense Oligonucleoitde-Based

PDE4 inhibitor drugs (Down et al. 2006).

would be an effective drug.

**3. PXS TPI1100: The drug** 

rather than the protein product.

second group target proteins.

**3.1 Antisense oligonucleotides: An overview** 

Therapy Directed at Phosphodiesterases 4 and 7 for COPD 439

Daxas (3-cyclopropylmethoxy-4-difluoromethoxy-*N*-[3,5-di-chloropyrid-4-yl]-benzamide) is a once-a-day tablet, taken orally, whose principal action is to reduce inflammation. The clinical results from the six Phase III trials performed using Daxas will be reviewed below. Before, touting the benefits of PDE4 inhibitors in COPD, it is important to note that Daxas is not without its adverse events which include diarrhea, weight loss, nausea, headache and abdominal pain (Giembycz &Field 2010), which have been observed previously with other

Like the PDE4 family, the PDE7 family, which consists of PDE7A and PDE7B, is highly selective for cAMP as a substrate (Bender &Beavo 2006). While the function of PDE7 has not been fully elucidated, PDE7 isoforms have been implicated in the activation of inflammatory cells (Li L et al. 1999), including T cells (Smith et al. 2003). PDE7A mRNA has been shown to be expressed in multiple tissues including the lung and inflammatory cells (Table 1) (Bloom &Beavo 1996) (Han et al. 1997) (Lugnier 2006). Inhibitors of PDE7 have shown to potentiate the effects of PDE4 inhibitors, suggesting that a combined PDE4:PDE7 inhibitor

The relative lack of advancement and the slow pace of innovation to identify new drug products for COPD can be indicative of the complicated nature of this chronic diseases as well as a potential limited number of targets for conventional small molecule drugs and biologics. Moreover, the activity of cytokines, growth factors and chemokines depends on the interaction of these proteins with their cell surface receptors involving large proteinprotein interactions or involving interactions between multiple sites on the protein, which could be particularly challenging to disrupt with small molecule inhibitors or biologics (Johnson et al. 2005). To side-step these complications, we have attempted to design an antisense oligonucleotide (AON) based therapy which functions by targeting RNA directly

Oligonucleotides (ODN) are short polymers of nucleotides that come in various forms, lengths and modifications which can be distinguished into two main groups based on two distinct mechanisms of action; ODN in the first group target RNA and those from the

RNA-targeting ODN drugs are designed to bind to a specific sequence of a messenger RNA (mRNA) through Watson-Crick base-pairing interactions. Therefore, the site of action of this class of drug is not the protein itself, but rather "upstream" of it, the RNA coding for the protein. The principle of RNA-based therapy is the reduction in the level of a protein through hindrance of its translation. Archetypes of this class of ODN are AON and small interfering RNA (siRNA). AON drugs are single stranded, usually only approximately 20 bases long, which prevent translation of the target RNA via one of two mechanisms. The first mechanism involves the activation of the enzyme RNAse H, which cleaves the RNA moiety of the duplex formed by the binding of the AON drug to its target RNA leading to subsequent reduction in protein synthesis (Stein &Hausen 1969). The second mechanism involves a steric interaction of the AON with the target mRNA that prevents key maturation

are reasons PDEs are good drug targets as selective inhibition of a specific PDE isoform would limit nonspecific sides effects associated with broader PDE inhibition.

Another reason PDEs have been the focus of drug companies is based on the pharmacologic principle that a more rapid and larger percentage change in concentration is achieved through regulating the degradation of a second messenger than comparable regulation of the rates of synthesis (Bender &Beavo 2006). In most cells the levels of cAMP are between <1 to 10 µM which enables a competitive inhibitor to not need to compete with high levels of endogenous substrate to be effective, in contrast to many protein kinase inhibitors which need to have sufficient affinity to displace mM concentrations of ATP (Bender &Beavo 2006).

There are four PDE4 (A/B/C/D) genes which generate multiple variants as a result of splicing differences in their N termini (Bender &Beavo 2006). PDE4 isoforms, which are widely expressed in many tissues and cell types including the lung, have been shown to play a key role in macrophage and monocyte activation and functions, neutrophils infiltration and vasodilation (Table 1). There has been more information collated on PDE4 than other PDEs mostly from the work resulting from PDE4A, 4B and 4D knock out mice. In PDE4D knockout mice, their airways were shown to be refractory to cholinergic stimulation (Mehats et al. 2003) while PDE4B knockout mice were shown to have effects on immune cells (Jin &Conti 2002; Jin et al. 2005) and both genes were shown to be required for neutrophils recruitment in a model of lung injury in response to inhaled endotoxin (Ariga et al. 2004).

A new first-in class treatment, the PDE4 inhibitor Daxas/Daliresp (Nycomed), has recently been approved in Europe in 2010 and in the USA in 2011 for patients with severe COPD.


Table 1. Expression of different cAMP-modulating PDE isoforms in lung cells and inflammatory cells. PDE4 and PDE7 are highly expressed in lung structural cells as well as in inflammatory cells. Delivered to the lung, PXS TPI1100 can inhibit expression of PDE4 and PDE7 in both lung structural and inflammatory cells.

Daxas (3-cyclopropylmethoxy-4-difluoromethoxy-*N*-[3,5-di-chloropyrid-4-yl]-benzamide) is a once-a-day tablet, taken orally, whose principal action is to reduce inflammation. The clinical results from the six Phase III trials performed using Daxas will be reviewed below. Before, touting the benefits of PDE4 inhibitors in COPD, it is important to note that Daxas is not without its adverse events which include diarrhea, weight loss, nausea, headache and abdominal pain (Giembycz &Field 2010), which have been observed previously with other PDE4 inhibitor drugs (Down et al. 2006).

Like the PDE4 family, the PDE7 family, which consists of PDE7A and PDE7B, is highly selective for cAMP as a substrate (Bender &Beavo 2006). While the function of PDE7 has not been fully elucidated, PDE7 isoforms have been implicated in the activation of inflammatory cells (Li L et al. 1999), including T cells (Smith et al. 2003). PDE7A mRNA has been shown to be expressed in multiple tissues including the lung and inflammatory cells (Table 1) (Bloom &Beavo 1996) (Han et al. 1997) (Lugnier 2006). Inhibitors of PDE7 have shown to potentiate the effects of PDE4 inhibitors, suggesting that a combined PDE4:PDE7 inhibitor would be an effective drug.
