Background
Atopic dermatitis (AD) is a common chronic inflammatory skin disease with substantial negative influence on the life quality of affected individuals. The central features of AD are skin dryness and eczematous lesions that are characterized by severe pruritus, erythema, excoriated/crusted papulovesicles, exudation and scaling, and in the chronic stage, skin thickening. The etiology of AD is complex and incompletely understood, but genetic predisposition in complex interplay with environmental factors is believed to be important [
1,
2].
Skin barrier dysfunction and aberrant immune responses are key factors in the pathogenesis of AD [
1,
3‐
5]. The acute stage of AD is typically dominated by a Th2-type of T cell response, believed to be triggered by environmental allergens [
1,
4,
6]. The cutaneous hyperreactivity is fueled by the dysfunctional, leaky skin barrier, which increases the exposure of the immune system for allergens [
1,
6,
7]. While the deficient skin barrier may amplify the cutaneous immune response, Th2 cytokines may also negatively influence the integrity of the epidermal barrier, e.g. by inhibiting the production of skin barrier proteins and antimicrobial peptides [
3,
4]. In its chronic stage, the AD lesion is characterized by an immune response involving several T cell subsets, with a significant contribution of the Th1 subset [
3,
6,
7]. This shift in T cell subset predominance may be caused by an increased exposure of the immune system to non-allergen triggers, including microbial products [
6]. In line with this, skin colonization with superantigen-producing strains of
S. aureus is very common in AD patients [
6,
8,
9].
With respect to pharmacological treatment of AD, the dysregulated immune response is still the process mainly targeted by available therapies, e.g. topical corticosteroids and calcineurin inhibitors [
2]. Although efficacious, side-effects and safety issues associated with these drugs limit their usefulness, especially as maintenance treatments. Thus, there is a need for new, safe anti-inflammatory/immunomodulatory agents that can be used for the induction and maintenance of clinical remission.
Several reports suggest that serotonin, i.e. 5-hydroxytryptamine (5-HT), first recognized as a vasoactive compound, plays a role in inflammation and immune responses as well as in pruritus, pain and fibrosis [
10‐
21], all processes of pathophysiological relevance in dermatitis. For instance, several animal studies suggest the importance of 5-HT and 5-HT
2 receptors for edema formation [
12,
14,
19], T cell responsiveness [
10,
16,
21] and scratching behaviour [
22‐
25]. In humans, intradermal injection of 5-HT causes erythema, edema and, importantly, pruritus with rapid onset [
20,
26]. In addition, 5-HT levels are increased in eczematous skin, e.g. in patients with allergic contact dermatitis (ACD) [
27,
28]. Moreover, the pro-inflammatory role of the 5-HT
2 receptor family in human skin is suggested by a clinical report showing the 5-HT
2 receptor antagonist ketanserin to reduce ACD, a T cell-dependent reaction [
29].
AM1030 is a novel 5-HT
2B receptor antagonist that displays binding to and functional inhibition of the human receptor (K
i = 0.33 μM; IC
50 = 0.14 μM). Structurally, AM1030 is an aminoguanidine derivative related to a previously published compound with anti-inflammatory properties [
30]. AM1030 is currently in clinical development phase.
Against this background, we initiated the current work, which had the objective to investigate AM1030 with respect to its anti-inflammatory profile and potential. To this end, a model-based approach was used, employing a set of distinct human and rodent in vitro and in vivo systems of relevance for a range of inflammatory diseases, including AD and ACD.
Discussion
In the current report a set of different in vitro and in vivo model systems was used to investigate the 5-HT2B receptor antagonist AM1030 with respect to its anti-inflammatory potential and profile. Based on the diversity of the employed model systems, e.g. with respect to activating triggers (SEA, LPS, recall antigen), responding cell populations (T cells, APCs, monocytes, macrophages), species (human, mouse and rat), read-outs (T cell cytokines, APC/monocyte/macrophage cytokines, in vivo inflammatory reactions) and drug administration routes (peroral, subcutaneous, topical), we conclude that AM1030 has broad anti-inflammatory and immunomodulatory effects.
The use of human PBMCs in combination with SEA enables us to mimic certain aspects of antigen presentation [
32], a process of general significance in inflammatory diseases, including AD. In this in vitro system, AM1030 was demonstrated to reduce the production of cytokines from both adaptive (IL-2, IFN-ɣ, IL-5) and innate (IL-12) immune cells (Fig.
1). Our initial observations in human PBMCs were corroborated by in vivo findings in mice, in which AM1030 was able to suppress the SEA-induced cytokines of interest (Fig.
3b-
e). The use of SEA as stimulus in our models substantiates the relevance of our findings in relation to AD, since AD patients are commonly colonized with superantigen-producing strains of
S. aureus [
8,
9]. By using LPS, a T cell-independent stimulus, in vitro and in vivo, we were then able to show the effect of AM1030 on monocyte/macrophage responses (Figs.
2 and
3a).
In summary, our combined pharmacological data and 5-HT
2B receptor expression data (mRNA; Table
1) suggest that the immunomodulatory effect of AM1030 in T cell-dependent reactions depends on its primary influence on cell types within the APC subset, such as monocytes and macrophages. The mechanistic basis for this influence remains to be investigated. Importantly, our results suggest that 5-HT
2B receptor signaling has impact on cytokine production by APCs, triggered by both MHC class II (SEA) and toll-like receptor 4 (LPS) stimulation. Therefore, follow-up studies should focus on identifying common denominators in intracellular signaling pathways shared by the 5-HT
2B receptor, MHC class II and toll-like receptor 4. The extracellular signal-regulated kinase (Erk) pathway is a possible candidate and might be a good starting point for such investigations, since it has been described to be involved in both 5-HT
2B receptor-induced signaling [
40] and LPS-induced pro-inflammatory cytokine production in monocytes [
41,
42]. Moreover, MHC class II activation has been shown to induce sustained Erk activity in APCs [
43]. With regard to the role of Erk in LPS-induced cytokine responses, our own unpublished data clearly show that inhibition of the Erk pathway with two distinct MEK (Erk kinase) inhibitors reduces TNF and IL-6 production in LPS-stimulated THP-1 cells. Follow-up studies should also address whether the 5-HT
2B receptor might regulate gene transcription independently from additional triggers.
The indirect effect of 5-HT
2B receptor activation on T cell responses that is suggested by our data should also be further addressed in future studies. Currently, one plausible explanation supported by the literature is that APC-derived pro-inflammatory cytokines, e.g. TNF, IL-6 and IL-12, can provide additional signals to enhance T cell activation [
44,
45].
The list of pathogenic mediators in AD and candidate target molecules for disease intervention is continuously growing. There are currently a few biological approaches to treatment underway that hold promise for the future, e.g. dupilumab, targeting the shared IL-4Rα subunit, thus interfering with both IL-4 and IL-13 signaling [
46], and ustekinumab, targeting the p40 subunit shared by IL-12 and IL-23 [
47,
48]. However, previous attempts to neutralize single mediators in AD have been rather disappointing, e.g. anti-IL-5 [
49] and anti-IgE [
50,
51], presumably due to the redundancy of disease-driving mediators combined with the heterogeneous nature of the disease. An alternative approach towards new treatment is the development of small molecular drugs that, rather than neutralizing a single mediator, have potential to simultaneously target several different pathogenic cell types and associated downstream processes. The relevance of this approach is shown by the well-established efficacy of topical corticosteroids and calcineurin inhibitors, having in common their broad immunomodulatory and anti-inflammatory effects [
2].
The anti-inflammatory and immunomodulatory effects of AM1030 and RS127445 reported herein support the concept of using 5-HT
2B receptor antagonists for the treatment of inflammatory diseases, including AD. This concept is also supported by earlier studies investigating the role of 5-HT and the 5-HT
2 receptor family in human and animal systems, addressing various aspects of inflammation, including pruritus [
10‐
27,
29]. Whether a rapid onset anti-pruritic effect could be achieved by topical administration of a 5-HT
2B receptor antagonist is currently unknown and beyond the scope of this paper. However, having in mind the questioned efficacy of anti-histamines in AD [
1], a 5-HT
2B receptor antagonist drug with rapid, yet sustained anti-pruritic and anti-inflammatory effects would be a welcome addition to the current treatment arsenal. Studies in humans will of course be necessary to explore the full potential of 5-HT
2B receptor antagonists in pruritic dermatitis.
The presented in vivo results show that AM1030, a 5-HT
2B receptor antagonist, has broad anti-inflammatory/immunomodulatory effects after systemic administration in different animal models (Figs.
3 and
4). However, in the course of our work, new information obtained from pharmacokinetic studies indicated a rapid systemic elimination of AM1030, which made us consider non-systemic routes of administration, such as topical application onto the skin. Interestingly, topically applied AM1030 reduced the oxazolone-induced DTH reaction in mice (Fig.
5), a T cell-dependent type IV hypersensitivity reaction with mechanistic resemblance to ACD as well as AD [
39]. Considering the reported ability of the 5-HT
2 receptor antagonist ketanserin to reduce ACD in humans [
29], our DTH results seem rather encouraging.