Apremilast, a novel PDE4 inhibitor, inhibits spontaneous production of tumour necrosis factor-alpha from human rheumatoid synovial cells and ameliorates experimental arthritis

Abstract

Introduction: Type 4 phosphodiesterases (PDE4) play an important role in immune cells through the hydrolysis of the second messenger, cAMP. Inhibition of PDE4 has previously been shown to suppress immune and inflammatory responses, demonstrating PDE4 to be a valid therapeutic target for immune-mediated pathologies. We assessed the anti-inflammatory effects of a novel PDE4 inhibitor, apremilast, in human synovial cells from rheumatoid arthritis (RA) patients, as well as two murine models of arthritis.

Methods: Cells liberated from tissue excised from arthritic joints of RA patients were cultured in the presence of increasing concentrations of apremilast for 48 hours and spontaneous tumour necrosis factor-alpha (TNFalpha) production was analysed in culture supernatants by ELISA. In addition, arthritis was induced in BALB/c and DBA/1 mice by passive transfer of anti-type II collagen mAb and immunisation with type II collagen, respectively. Mice with established arthritis received 5 or 25 mg/kg apremilast and disease severity was monitored relative to mice receiving vehicle alone. At the end of the study, paws were removed and processed for histopathological assessment. Behavioural effects of apremilast, relative to rolipram, were assessed in naïve DBA/1 mice using an automated activity monitor (LABORAS).

Results: Apremilast dose dependently inhibited spontaneous release of TNFalpha from human rheumatoid synovial membrane cultures. Furthermore, apremilast significantly reduced clinical score in both murine models of arthritis over a ten day treatment period and maintained a healthy joint architecture in a dose-dependent manner. Importantly, unlike rolipram, apremilast demonstrated no adverse behavioural effects in naïve mice.

Conclusions: Apremilast is an orally available PDE4 inhibitor that reduces TNFalpha production from human synovial cells and significantly suppresses experimental arthritis. Apremilast appears to be a potential new agent for the treatment of rheumatoid arthritis.

Figure 1

Apremilast inhibits TNFα release from LPS-stimulated monocytes and human rheumatoid synovial membrane cultures. Human peripheral blood monocytes were treated with increasing concentrations of apremilast, 30 minutes before stimulating with 10 ng/ml lipopolysaccharide (LPS) for 24 hours. (a) Culture supernatants were assayed for TNFα by ELISA. Data is representative of three donors. Human rheumatoid arthritis (RA) synovial membrane cells were cultured for 48 hours in the presence of apremilast, rolipram or controls and spontaneous production of (b) TNFα, (c) IL-6 and (d) IL-10 in culture supernatants was measured by ELISA. Means and standard errors of percent inhibition, from five (apremilast) or two (rolipram) donors are plotted. As a positive control, cells were treated with a combination of anti-TNFα mAb and IL-1RA. Statistical analysis was calculated by one-way analysis of variance and Dunnett's multiple comparison test. Each test group was compared to (a) cells + LPS, or (b to d) cells only. * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 2

Apremilast reduces severity of mAb-induced arthritis in BALB/c mice. (a) Arthritic mice were treated orally with apremilast, dexamethasone or vehicle and disease severity was evaluated and assigned a clinical score. (b) Area under the curve for each mouse was calculated and statistical significance relative to vehicle control group was analysed (*** P < 0.001). (c) Paw thickness was measured throughout with microcalipers and statistical significance was calculated by two-way analysis of variance with Bonferroni post test analysis. Means and standard error of the mean are plotted, eight mice per group (*** P < 0.001). H&E staining of a longitudinal section through the tibio-tarsal joint from a vehicle-treated mouse and one treated with 25 mg/kg apremilast is shown in (d). Asterisk shows inflammation of the synovial membrane, small arrow, erosion of articular cartilage, and bold arrow, fibrin deposits and inflammatory cell infiltrate within the articular cavity. Images were acquired at 100 × magnification.

Figure 3

Apremilast reduces severity of collagen-induced arthritis in DBA/1 mice. Arthritic, male DBA/1 mice were treated from day one of onset to day 10 post onset of arthritis with a daily, intra-peritoneal dose of apremilast or vehicle, after which the mice were sacrificed and paws were removed for histological analysis. Disease severity was evaluated throughout. Change in clinical score is plotted in (a). Each treatment group comprised of 14 mice, with means and standard errors plotted. (b) The statistical significance of the clinical score of apremilast-treated compared with vehicle-treated mice was calculated from area under the curve (AUC) for each mouse over the 10 day treatment period (* P < 0.05). Representative images of H&E-stained sections through the proximal interphalangeal joint of an apremilast-treated mouse and a vehicle-treated mouse are shown (c). Scale bars are 500 μm. (d) All sections were scored for the extent of inflammation and damage, and graded accordingly. (* P < 0.05).

Figure 4

The effect of 25 mg/kg apremilast or rolipram on spontaneous behaviour was tested on naïve mice using LABORAS automated activity monitor. The average time spent (a) immobile and grooming and (b) climbing and during locomotion are plotted. n = 8 per group, * P < 0.05, *** P < 0.001, relative to vehicle treatment. Mice were monitored for 30 minutes, one hour after receiving treatment. n.s., not significant.

Figure 5

Apremilast inhibits antigen specific T cell cytokine production and proliferation in lymph node cultures. Male DBA/1 mice were immunised with bCII in CFA and 14 days later, inguinal lymph nodes were removed and cells were dissociated. Lymph node cultures, unstimulated, or stimulated with either bCII or anti-CD3 mAb, were cultured in the presence of increasing concentrations of apremilast or 100 μM rolipram for 48 hours at which time supernatants were removed and analysed for (a) TNFα, and (b) interferon (IFN)γ production by ELISA. (c) Cells were then pulsed with ³H- thymidine for a further 20 hours to assess cell proliferation. Statistical significance between treatment and control groups was calculated by two-way analysis of variance with Bonferroni multiple comparison test. (* P < 0.05, *** P < 0.001).