Abstract
IntroductionThis work was undertaken to delineate intracellular signaling pathways for the PDE4 inhibitor apremilast and to examine interactions between apremilast, methotrexate and adenosine A2A receptors (A2AR).MethodsAfter apremilast and LPS incubation, intracellular cAMP, TNF-α, IL-10, IL-6 and IL-1α were measured in the Raw264.7 monocytic murine cell line. PKA, Epac1/2 (signaling intermediates for cAMP) and A2AR knockdowns were performed by shRNA transfection and interactions with A2AR and A2BR, as well as with methotrexate were tested in vitro and in the murine air pouch model. Statistical differences were determined using one or two-way ANOVA or Student’s t test. The alpha nominal level was set at 0.05 in all cases. A P value of < 0.05 was considered significant.ResultsIn vitro, apremilast increased intracellular cAMP and inhibited TNF-α release (IC50=104nM) and the specific A2AR-agonist CGS21680 (1μM) increased apremilast potency (IC50=25nM). In this cell line, apremilast increased IL-10 production. PKA, Epac1 and Epac2 knockdowns prevented TNF-α inhibition and IL-10 stimulation by apremilast. In the murine air pouch model, both apremilast and MTX significantly inhibited leukocyte infiltration, while apremilast, but not MTX, significantly inhibited TNF-α release. The addition of MTX (1 mg/kg) to apremilast (5 mg/kg) yielded no more inhibition of leukocyte infiltration or TNF-α release than with apremilast alone.ConclusionsThe immunoregulatory effects of apremilast appear to be mediated by cAMP through the downstream effectors PKA, Epac1, and Epac2. A2AR agonism potentiated TNF-α inhibition by apremilast, consistent with the cAMP-elevating effects of that receptor. Because the A2AR is also involved in the anti-inflammatory effects of MTX, the mechanism of action of both drugs involves cAMP-dependent pathways and is therefore partially overlapping in nature.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0771-6) contains supplementary material, which is available to authorized users.
Highlights
This work was undertaken to delineate intracellular signaling pathways for the phosphiodiesterase type 4 (PDE4) inhibitor apremilast and to examine interactions between apremilast, methotrexate and adenosine A2A receptors (A2AR)
PDE inhibitors such as theophylline were being demonstrated during the 1970s [3]. cyclic adenosine monophosphate (cAMP)-specific phosphiodiesterase type 4 (PDE4), a critical regulator of intracellular cAMP levels and compartmentalization [4], is mainly expressed within inflammatory cells [5]
In the air pouch model, an in vivo model that mimics the synovial cavity, we have demonstrated that the antiinflammatory actions of MTX, the cornerstone treatment for rheumatoid arthritis, are mediated in large part, by increasing adenosine levels [17], which, via activation of the A2AR, increase intracellular cAMP levels [21]
Summary
This work was undertaken to delineate intracellular signaling pathways for the PDE4 inhibitor apremilast and to examine interactions between apremilast, methotrexate and adenosine A2A receptors (A2AR). Cyclic AMP-specific phosphodiesterases (PDE), have emerged as a new target for the treatment of numerous inflammatory conditions (reviewed in [2]) as the anti-inflammatory properties of PDE inhibitors such as theophylline were being demonstrated during the 1970s [3]. CAMP-specific phosphiodiesterase type 4 (PDE4), a critical regulator of intracellular cAMP levels and compartmentalization [4], is mainly expressed within inflammatory cells [5]. The clinical development of a class of PDE4 inhibitor anti-inflammatory agents has been hindered by their side effects, principally nausea and emesis [10]. Despite their potential as anti-inflammatory agents, PDE4
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