In vitro and in vivo anti-inflammatory effects of Dissociated Glucocorticoids Receptors Agonists (DGRA).

Abstract

Purpose: Chronic use of steroidal Glucocorticoid (GC) agonists to treat ocular inflammatory diseases results in increased ocular pressure leading to glaucoma.<br /> We aimed at identifying dissociated Glucocorticoids Receptors Agonists (DGRA) that could elicit a reduced transactivation (TA) response, while being able to maintain a potent transrepression (TR) of pro-inflammatory genes.

Methods: Binding assay: Binding to the GC receptor was evaluated using GC receptor Competitor assay (Panvera) using fluorescent probe (Fluormone ^TM GS Red).<br /> IL-6 release : Cells were treated with SAR1 and stimulated with IL-1b (1ng/mL). 24h later, cell number was counted using the CellTiter-Glo assay kit. The amount of IL-6 was quantified in the supernatant. The IL-6 levels were normalized to the total cell number.<br /> TR/TA assay: A549 cells were plated in DMEM with 10% FBS and incubated overnight (37°C; 5% CO₂). 60 min after treatment, cells were stimulated with IL-1b (1ng/mL). 5 hours post stimulation cells were lysed and the lysate was used to quantitate mRNA levels of IL-8 and TSC22D3 transcripts by TaqMan RT-PCR.<br /> Ovalbumin-induced allergic conjunctivitis in guinea pigs: 14 days after sensitization with ovalbumin (OVA 1mL/100µg/mL i.p.) allergic reaction was induced by topical OVA (30µL/100mg/L). 30 and 90 min after OVA challenge, treatments were topically applied and compared to vehicle (PEG3350/PS80/PBS)(1/10/89). Clinical signs were scored 3 and 6 h later.

Results: SAR1 bound to the GC receptor with a value of 1 nM and in the IL-6 release assay, SAR1 displayed anti-inflammatory effect with an IC₅₀ of 2.2nM and an efficacy of 92% compared to dexamethasone.<br /> In IL-1b stimulated A549 cells, SAR1 elicited a TR response against IL-8 with an IC₅₀ of 2.4 nM (efficacy 89%) and a TA response for TSC22D3 induction with an EC₅₀ of 16 nM (51% efficacy vs dexamethasone).<br /> Topically administered at 0.5%, SAR1 decreased significantly total clinical score up to 6h after the OVA-challenge in comparison to vehicle (-111%; p<0.005). These effects were found to be identical to those observed with betamethasone (-120%; p<0.001).

Conclusions: We have identified a potent and dissociated glucocorticoid agonist displaying in vivo activity in an allergic model of conjunctivitis. The next step will consist in testing DGRA compounds in a model of GC-induced ocular hypertension in rat.