Background/Purpose Phosphodiesterase 4 (PDE4) inhibitors have clear immunoregulatory effects in laboratory studies, and the clinical application of this class of drug in the field of rheumatology is now beginning with the introduction of the novel PDE4 inhibitor apremilast, which was recently approved by the FDA for the treatment of psoriatic arthritis. Although apremilast is known to increase intracellular cAMP levels, less is known about its downstream signaling mediators. We therefore undertook this work to delineate intracellular signaling pathways for apremilast and to examine interactions between apremilast, methotrexate (MTX), and adenosine A2A receptors (A_2AR).

Methods After apremilast and lipopolysaccharide incubation, intracellular cAMP, TNF-α, IL-10, IL-6, and IL-1α were measured in the Raw264.7 mouse monocytic cell line. PKA, Epac1/2 (signaling intermediates for cAMP), and A₂R knockdowns were performed by shRNA transfection and interactions with A_2AR and A_2BR. Apremilast and MTX were tested in vivo in the murine air pouch model.

Results In vitro, apremilast increased intracellular cAMP (apremilast 1 μM: 240.0±57.7% of control, n=3, P<0.001) and reduced TNF-α release (pIC₅₀=6.98±0.20, n=8), and MTX reduced TNF-α release (maximum inhibition 50.9±10.2% of vehicle, n=3). The specific A_2AR-agonist CGS21680 (1 μM) increased apremilast potency (pIC₅₀=7.59±0.20, n=8), suggesting that apremilast-mediated TNF-α inhibition can be enhanced by signaling through A_2AR. In this mouse-derived cell line, apremilast increased IL-10 (apremilast 100 nM: 576.3±32.8% of control, n=3, P<0.001) and IL-6 (apremilast 100 nM: 257±53.1% of control, n=3, P<0.05). PKA, Epac1, and Epac2 knockdowns reduced 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 (apremilast 5 mg/kg: leukocyte accumulation 72.5±11.6% of vehicle, n=6, P<0.05; TNF-α release 65.5±7.7% of vehicle, n=6, P<0.001; MTX 1 mg/kg: leukocyte accumulation 54.6±3.9% of vehicle, n=20, P<0.001; TNF-α release 83.7±8.9% of vehicle, n=5, P>0.05). The addition of MTX (1 mg/kg) to apremilast (5 mg/kg) yielded no more inhibition of leukocyte infiltration or TNF-α release than that of apremilast alone.

Conclusion The immunoregulatory effects of apremilast appear to be mediated by cAMP through the downstream effectors PKA, Epac1, and Epac2. A_2AR agonism potentiated TNF-α inhibition by apremilast, consistent with the cAMP-elevating effects of that receptor. Because A_2AR 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.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/apremilast-a-novel-phosphodiesterase-4-inhibitor-and-methotrexate-independently-prevent-inflammation-in-vivo-and-in-vitro/