The PDE4 inhibitor CHF6001 affects keratinocyte proliferation via cellular redox pathways
Introduction
Psoriasis is a chronic, inflammatory skin disease that affects 1%–3% of the population worldwide and involves keratinocyte hyperproliferation in the epidermis, inflammatory infiltrates, and aberrant differentiation [1]. The phenotypic appearance of psoriasis is characterized by red lesions covered by silvery scale-like plaques. The intensity of psoriatic lesions can vary from a few random spots to a massive outbreak that covers the entire body. There is no cure for this disease; treatments are primarily focused on reducing or clearing plaques as well as preventing remission by modulating inflammation and slowing down psoriasis-associated rapid cell growth and shedding.
In recent years, an improved understanding of the pathomechanisms underlying this disease has lead to the developments of new treatments. Recent studies showed that in psoriatic patients, expression of all phosphodiesterase 4 (PDE4) isoforms (A-D), part of a family of enzymes known to regulate cyclic adenosine monophosphate (cAMP) levels and immune homeostasis, is elevated compared to healthy controls [2]. cAMP is a second messenger involved in regulating a wide array of immune-related genes such as IL-2, IL-6, IL-10, and Tumor Necrosis Factor alpha (TNFα) through cAMP-responsive elements located in gene promoters, as well as through crosstalk with Nuclear Factor-κB (NF-κB) signaling [3]. Several PDE4 inhibitors have recently been evaluated in clinical trials for treating various skin inflammatory conditions, including apremilast, roflumilast, and crisabolore, with apremilast approved for the treatment of psoriasis and crisaborole for the treatment of atopic dermatitis [4]. In particular, aprelimilast exhibits pleiotropic, synergistic, and attenuating effects on a key group of cytokines involved in the pathogenesis of psoriasis, most notably IL-17A/F, IL-22, and TNF-α, and these effects correlate with reduced skin manifestations of the disease [2]. However, common target-related adverse events including diarrhea, nausea, and headache are associated with apremilast usage [4]. Therefore, alternative routes of administration could be explored with the goal of increasing tolerability by utilizing drugs designed for topical treatment and reducing systemic effects.
CHF6001 is an inhaled selective inhibitor of PDE4 isoforms A-D [5], which is endowed with anti-inflammatory properties in several in vitro models, involving lymphocytes, monocytes, macrophages, and dendritic cells [[6], [7], [8], [9], [10], [11]]. Clinical studies have shown that topical administration of CHF6001 is well-tolerated [12] and reduces the allergen challenge response in asthmatic patients [13]. CHF6001 is currently in clinical development for the treatment of Chronic Obstructive Pulmonary Disease (COPD) (Clinical trials.gov Identifier NCT02986321).
In the present study, we investigated whether the PDE4 inhibitor CHF6001 could be used as an agent to modulate proliferation and inflammation in keratinocytes in vitro. Since psoriasis is characterized by distinct histological features of the skin, including keratinocyte hyperproliferation, we evaluated the effect of CHF6001 on keratinocyte (HaCaT) proliferation and re-epithelialization in a scratch-wound model, along with crucial molecules involved in cell cycle progression, i.e. cyclin D1, and key players in redox-inflammatory crosstalk, such as NF-κB and NADPH oxidase.
Section snippets
Cell culture and treatments
HaCaT keratinocytes (a gift from Dr. F. Virgili), were grown in Dulbecco's modified Eagle's medium High Glucose (Lonza, Milan, Italy) at 37 °C in 95%air/5%CO2, as previously described by Sticozzi et al., 2012 [16].
HaCaT cells were treated with different doses (0.1–10 μM) of the specific PDE4 inhibitor CHF6001 for 24 h. This compound was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM as a stock solution. The stock was then diluted to the required concentrations directly in
Effect of CHF6001 on cellular toxicity
The first step of the present study was to determine whether CHF6001 affected keratinocyte (HaCaT) viability. Cellular cytotoxicity of CHF6001 in concentrations ranging from 0.1 to 10,000 nM was analysed by using an LDH release assay. As shown in Fig. 1, after 24 h of treatment with CHF6001, no cytotoxic effect was detected in HaCaT cells, with respect to control (DMSO 0.1%). Cells treated with Triton X-100 represent the positive control (i.e. 100% of mortality) (Fig. 1).
Effect of CHF6001 on cellular proliferation
Based on our viability
Discussion
Our study provides a rationale for testing the effects of topical application of PDE4 inhibitor CHF6001 in inflammatory skin disorders, such as psoriasis. Indeed, the rationale for the use of PDE4 inhibitors for treatment of psoriasis has been recently reinforced by the approval of the oral PDE4 inhibitor apremilast for moderate-to-severe plaque psoriasis. The mechanism of action of apremilast has been associated with its anti-inflammatory properties [39].
First, we performed cellular viability
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