Anti-inflammatory and immunosuppressive properties of the novel CB1 receptor ligand E-6776

Miguel Angel Iñiguez1, Carmen Punzón1, Ruth Álvarez1, Sergio Ovalle2, Helmut Buschmann2, José Miguel Vela2, Manuel Fresno1. 1Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Madrid, Spain, 2Laboratorios Esteve, S.A., Barcelona, Spain.

Recent evidence supports the potential immunomodulatory and anti-inflammatory properties of cannabinoid-based drugs. In this way, not only agonists (Croxford and Yamamura, 2005) but also antagonists (Smith et al., 2000; Costa et al., 2005; Roche et al., 2006) have been described to exhibit anti-inflammatory activities.

On this basis, we have analyzed the effects of a new cannabinoid-based drug, the CB1 receptor antagonist E-6776 ((RS)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-N-(piperidin-1-yl)-4,5-dihydro-1H-pyrazole-3-carboxamide), as regulator of the immune response and inflammation, through the evaluation of its influence on different parameters of T cell and macrophage activation. We have used two human cellular models: Jurkat T cells stimulated with the phorbol ester TPA plus the calcium ionophore A23187 (Ion), and THP-1 macrophages stimulated with LPS. Reporter gene assays were used to evaluate the transcriptional modulation of the production of cytokines and other inflammatory mediators (Iñiguez et al., 1999, 2000). Data shown are the mean of triplicate results of at least two independent experiments. Statistical significance was analyzed using Student’s t test. IC50 values were derived by nonlinear curve-fitting of dose-response curves.

The CB1 antagonist E-6776 was able to inhibit cell proliferation in TPA+Ion activated T cells and in LPS-activated macrophages. E-6776 was also able to inhibit production of cytokines such as Interleukin (IL)-2 (IC50 6μM) and Tumor Necrosis Factor (TNF) alpha (IC50 6μM) in TPA+Ion stimulated Jurkat T cells. This effect was mainly due to its ability to modulate transcription as E-6776 treatment significantly (p<0.05) inhibited activation-dependent transcriptional induction of IL-2 (IC50 1μM), TNFalpha (IC50 1μM), IL-6 (IC50 4μM), inducible Nitric Oxide Synthase (iNOS) (IC50 4μM) and cyclooxygenase (COX)-2 (IC50 8μM) promoters in a concentration-dependent manner. Interestingly, this CB1 ligand was able to inhibit significantly the transcription-dependent activation of the Nuclear Factor of Activated T cells (NFAT) (IC50 3μM) and Nuclear Factor (NF)-kappaB (IC50 7μM). NFAT dephosphorylation and nuclear translocation, an essential step in NFAT activation, was also affected by this compound.

These results may have important implications in anti-inflammatory therapy and open a potentially new field for cannabinoid-based drugs as modulators of immune activation and the inflammatory response.

Costa et al. (2005) Pain 116:52-61

Croxford and Yamamura (2005) Neuroimmunol 166:3-18

Iñiguez et al. (1999) J Immunol 163:111-9

Iñiguez et al. (2000) J Biol Chem 275:23627-35

Roche et al. (2006) J Neuroimmunol 181:57-67

Smith et al. (2000) J Pharmacol Exp Ther 293:136-50.