Adelmidrol increases the endogenous concentrations of palmitoylethanolamide in canine keratinocytes and down-regulates an inflammatory reaction in an in vitro model of contact allergic dermatitis

Highlights

• The mechanism of action of adelmidrol was studied.

• Adelmidrol increases PEA and 2-AG levels in canine keratinocytes.

• Adelmidrol is not an inhibitor of PEA catabolic enzymes, NAAA and FAAH.

• Adelmidrol is not a cytotoxic compound.

• Adelmidrol inhibits an inflammatory reaction in stimulated keratinocytes.

Abstract

This study aimed to investigate potential new target(s)/mechanism(s) for the palmitoylethanolamide (PEA) analogue, adelmidrol, and its role in an in vitro model of contact allergic dermatitis. Freshly isolated canine keratinocytes, human keratinocyte (HaCaT) cells and human embryonic kidney (HEK)-293 cells, wild-type or transfected with cDNA encoding for N-acylethanolamine-hydrolysing acid amidase (NAAA), were treated with adelmidrol or azelaic acid, and the concentrations of endocannabinoids (anandamide and 2-arachidonoylglycerol) and related mediators (PEA and oleoylethanolamide) were measured. The mRNA expression of PEA catabolic enzymes (NAAA and fatty acid amide hydrolase, FAAH), and biosynthetic enzymes (N-acyl phosphatidylethanolamine-specific phospholipase D, NAPE-PLD) and glycerophosphodiester phosphodiesterase 1, was also measured. Brain or HEK-293 cell membrane fractions were used to assess the ability of adelmidrol to inhibit FAAH and NAAA activity, respectively. HaCaT cells were stimulated with polyinosinic–polycytidylic acid and the release of the pro-inflammatory chemokine, monocyte chemotactic protein-2 (MCP-2), was measured in the presence of adelmidrol.

Adelmidrol increased PEA concentrations in canine keratinocytes and in the other cellular systems studied. It did not inhibit the activity of PEA catabolic enzymes, although it reduced their mRNA expression in some cell types. Adelmidrol modulated the expression of PEA biosynthetic enzyme, NAPE-PLD, in HaCaT cells, and inhibited the release of the pro-inflammatory chemokine MCP-2 from stimulated HaCaT cells. This study demonstrates for the first time an ‘entourage effect’ of adelmidrol on PEA concentrations in keratinocytes and suggests that this effect might mediate, at least in part, the anti-inflammatory effects of this compound in veterinary practice.

Introduction

Chronic allergic skin diseases represent a growing burden, and more than 20% of the animals are referred to a veterinary doctor for a dermatological problem (Hill et al., 2006). Although topical anti-inflammatory treatments are considered the reference standard for managing focal or multifocal skin lesions in atopic dermatitis (Olivry et al., 2010), only few alternatives to topical steroids are available. The search for valuable treatment options to be safely applied for long durations has recently focused on the cutaneous endocannabinoid system (Lambert, 2007, Biro et al, 2009, Kupczyk et al, 2009), including cannabinoid receptors, the endocannabinoids (anandamide and 2-arachidonoylglycerol), structurally similar bioactive amides (such as palmitoylethanolamide, PEA), and their biosynthetic and catabolic enzymes (Fezza and Maccarrone, 2014).

The main physiological function of the endocannabinoid system in the skin is to preserve the local homeostasis and the complex cellular dynamics (Biro et al., 2009). Recent studies have suggested the existence of the endocannabinoid system in the canine skin (Campora et al., 2012) and it has been shown that PEA concentrations in the skin change during canine atopic dermatitis (Abramo et al., 2014). Moreover, PEA was recently found to afford significant therapeutic benefit in dogs with experimental hypersensitivity (Cerrato et al., 2012a) and spontaneous atopic dermatitis (Noli et al., 2014).

Adelmidrol is an analogue of PEA. Unlike PEA, which is highly lipophilic, adelmidrol is suitable for topical application because it exhibits both hydrophilic and lipophilic features (Cerrato et al., 2012b). Adelmidrol is the di-ethanolamide derivative of azelaic acid, a natural substance, topically effective for human inflammatory skin disorders (Nazzaro-Porro, 1987) and able to modulate the inflammatory response in human keratinocytes (Mastrofrancesco et al., 2010).

Several mechanisms of action have been proposed for PEA (Petrosino et al., 2010a), including: (1) the direct stimulation of an as-yet uncharacterised cannabinoid type 2-like receptor (Conti et al, 2002, Farquhar-Smith, Rice, 2003); (2) an ‘entourage effect’ (Di Marzo et al, 2001, Petrosino et al, 2015); and (3) the direct stimulation of particular molecular targets (Lo Verme et al., 2005). By contrast, the mechanism of action of adelmidrol has been investigated only in part (De Filippis et al., 2009). Yet, topical application of adelmidrol alleviates chronic inflammatory skin conditions in both humans (Pulvirenti et al., 2007) and dogs (Mantis et al, 2007, Cerrato et al, 2012b, Fabbrini, Leone, 2013).

The aim of the present study was to investigate the mechanism(s) of action of adelmidrol and its potential effect in an in vitro model of contact allergic dermatitis (CAD).