Apremilast, a cAMP phosphodiesterase-4 inhibitor, demonstrates anti-inflammatory activity in vitro and in a model of psoriasis

Abstract

Background and purpose: Apremilast is an orally administered phosphodiesterase-4 inhibitor, currently in phase 2 clinical studies of psoriasis and other chronic inflammatory diseases. The inhibitory effects of apremilast on pro-inflammatory responses of human primary peripheral blood mononuclear cells (PBMC), polymorphonuclear cells, natural killer (NK) cells and epidermal keratinocytes were explored in vitro, and in a preclinical model of psoriasis.

Experimental approach: Apremilast was tested in vitro against endotoxin- and superantigen-stimulated PBMC, bacterial peptide and zymosan-stimulated polymorphonuclear cells, immunonoglobulin and cytokine-stimulated NK cells, and ultraviolet B light-activated keratinocytes. Apremilast was orally administered to beige-severe combined immunodeficient mice, xenotransplanted with normal human skin and triggered with human psoriatic NK cells. Epidermal skin thickness, proliferation index and inflammation markers were analysed.

Key results: Apremilast inhibited PBMC production of the chemokines CXCL9 and CXCL10, cytokines interferon-gamma and tumour necrosis factor (TNF)-alpha, and interleukins (IL)-2, IL-12 and IL-23. Production of TNF-alpha by NK cells and keratinocytes was also inhibited. In vivo, apremilast significantly reduced epidermal thickness and proliferation, decreased the general histopathological appearance of psoriasiform features and reduced expression of TNF-alpha, human leukocyte antigen-DR and intercellular adhesion molecule-1 in the lesioned skin.

Conclusions and implications: Apremilast displayed a broad pattern of anti-inflammatory activity in a variety of cell types and decreased the incidence and severity of a psoriasiform response in vivo. Inhibition of TNF-alpha, IL-12 and IL-23 production, as well as NK and keratinocyte responses by this phosphodiesterase-4 inhibitor suggests a novel approach to the treatment of psoriasis.

Figure 1

Chemical structure of apremilast, (S)-N-{2-[1-(3-ethoxy-4-methoxyphenyl)-2-methanesulphonylethyl]-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl}acetamide.

Figure 2

(A) Time course of pro-inflammatory cytokine mRNA expression in LPS-stimulated PBMC. PBMC were either not stimulated (time = 0) or stimulated with LPS for 2, 4, 8 and 16 h. Cytokine mRNA levels were measured by RT-PCR and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels. All cytokine mRNAs at time zero were assigned a value of one. Data represent the mean ± SEM from two to three experiments. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by one-way anova followed by Dunnett's multiple comparison test, comparing each mRNA level with its level at time 0. (B) Reduction of pro-inflammatory cytokine mRNA levels by apremilast. PBMC were treated with vehicle (open columns) or 10 μM apremilast (coloured columns) for 1 h prior to stimulation with LPS for the indicated duration to achieve the maximum mRNA expression for each particular cytokine. mRNA levels were measured by RT-PCR and normalized to GAPDH mRNA levels. Data represent the mean ± SEM from three experiments. **P < 0.01, ***P < 0.001, as determined by a repeated measures anova followed by a Bonferroni's post-test comparing each pair of groups. IFN-γ, interferon-γ; IL, interleukin; LPS, lipopolysaccharide; PBMC, peripheral blood mononuclear cells; TNF-α, tumour necrosis factor-α.

Figure 3

Effect of apremilast on cytokine and chemokine production by LPS-stimulated PBMC. (A) Inhibition of cytokines by apremilast. (B) Inhibition of chemokines by apremilast. Results are means from three experiments, except for IL-12p70 (n = 4). SEMs are not shown to improve graph readability, but were similar to the SEMs shown in (C). Inhibition of all cytokines and chemokines shown in (A) and (B) was statistically significant by one-way anova (P < 0.05). (C) Apremilast elevated IL-10 and IL-6 production and had no effect on IL-8, IL-1β and RANTES production. Data are mean ± SEM from three experiments, except for IL-10 and RANTES (n = 2). **P < 0.01, by one-way anova followed by Dunnett's multiple comparisons post-test, as compared with vehicle control cultures. GM-CSF, granulocyte macrophage-colony stimulating factor; IFN-γ, interferon-γ; IL, interleukin; LPS, lipopolysaccharide; MCP, monocyte chemoattractant protein; MIG, monokine induced by IFN-γ (CXCL9); MIP, macrophage inflammatory protein; PBMC, peripheral blood mononuclear cells; RANTES, regulated on activation, normal T cell expressed and secreted; TNF-α, tumour necrosis factor-α.

Figure 4

Inhibition of cytokine production by NK cells. Human NK cells from peripheral blood were stimulated with IL-2 only or with IL-2 plus IgG. Data shown are mean ± SEM from two experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way anova followed by Dunnett's multiple comparisons post-test, as compared with the IL-2 + IgG stimulated control samples. GM-CSF, granulocyte macrophage-colony stimulating factor; IFN-γ, interferon-γ; IL, interleukin; MIP, macrophage inflammatory protein; NK, natural killer; TNF-α, tumour necrosis factor-α.

Figure 5

Apremilast inhibited keratinocyte tumour necrosis factor (TNF)-α production but not proliferation or cell viability. (A) Normal human epidermal keratinocytes were treated with apremilast or cyclosporine A for 2 days and assayed for cell proliferation. Data are mean ± SEM from three experiments. **P < 0.01, ns, not significant, by one-way anova followed by Dunnett's multiple comparison test versus vehicle control. (B) Normal human epidermal keratinocytes were treated with apremilast or 0.1% dimethylsulphoxide as a control, prior to ultraviolet B irradiation to induce TNF-α production. Supernatants were analysed for TNF-α protein production by elisa. TNF data are mean ± SEM from three experiments. **P < 0.01 by one-way anova followed by Dunnett's multiple comparisons post-test, as compared with vehicle control group.

Figure 6

(A) Histological section of human skin graft on severe combined immunodeficient (SCID) mouse, injected with natural killer (NK)-like cells of psoriatic patient, exhibits histological parameters of psoriasis, as follows: parakeratosis, hyperkeratosis, collection of neutrophils (Munro micro-abscess formation), absence of the granular layer and a focal area of spongiosis with a lymphocytic infiltrate. Additionally, epidermal thickening (acanthosis) and elongation of the rete ridges are detected. Vascular dilatation associated with a perivascular inflammatory cell infiltrates is observed in the dermis. (B) Histological section of another human skin graft on SCID mouse, injected with NK-like cells of psoriatic patient, demonstrates parakeratosis, hyperkeratosis, absence of granular layer, hyperplasia and suprapapillary epidermal thinning. Oedema of the dermal papilla is seen combined with dilated, tortuous dermal blood vessel, surrounded by several lymphocytes and neutrophils. (C) Histological section of human skin graft on SCID mouse, injected with NK-like cells of psoriatic patient, shows a complete recovery of the psoriatic phenotype, following treatment with apremilast (5 mg·kg⁻¹·day⁻¹ divided into two daily oral doses). Bar length = 50 µm.

Figure 7

Apremilast reduced epidermal thickness and proliferation index in the psoriasiform xenograft model. (A) Epidermal thickness and (B) proliferation index in normal human skin xenotransplanted along with psoriatic patient NK cells into beige-severe combined immunodeficient (SCID) mice. Columns represent the mean ± SEM of seven beige-SCID mice. **P < 0.01, ***P < 0.001, one-way anova followed by Dunnett's multiple comparison test versus vehicle control.