The PDE4 inhibitor CHF6001 affects keratinocyte proliferation via cellular redox pathways

Abstract

Psoriasis is a skin disease characterized by abnormal keratinocyte proliferation and inflammation. Currently, there are no cures for this disease, so the goal of treatment is to decrease inflammation and slow down the associated rapid cell growth and shedding. Recent advances have led to the usage of phosphodiesterase 4 (PDE4) inhibitors for treatment of this condition. For example, apremilast is an oral, selective PDE4 inhibitor that is able to reduce skin inflammation and is Food and Drug Administration (FDA)-approved to treat adults with moderate to severe psoriasis and/or psoriatic arthritis. However, common target-related adverse events, including diarrhea, nausea, headache, and insomnia limit the usage of this drug. To circumvent these effects, the usage of PDE4 inhibitors specifically designed for topical treatment, such as CHF6001, may combine local anti-inflammatory activity with limited systemic exposure, improving tolerability. In this study, we showed that CHF6001, currently undergoing clinical development for COPD, suppresses human keratinocyte proliferation as assessed via BrdU incorporation. We also observed decreased re-epithelialization in a scratch-wound model after CHF6001 treatment. At the molecular level, CHF6001 inhibited translocation of phosphorylated NF-κB subunit p65, promoting loss of nuclear cyclin D1 accumulation and an increase of cell cycle inhibitor p21. Furthermore, CHF6001 decreased oxidative stress, measured by assessing lipid peroxidation (4-HNE adduct formation), through the inactivation of the NADPH oxidase.

These results suggest that CHF6001 has the potential to treat skin disorders associated with hyperproliferative keratinocytes, such as psoriasis by targeting oxidative stress, abnormal re-epithelization, and inflammation.

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.