Denufosol tetrasodium

Denufosol tetrasodium (INS37217) is a long-acting P2Y2 receptor agonist, which exhibits an EC50 of ~10 μM for P2Y2 receptor activation[1].

Signaling Pathways >> GPCR/G Protein >> P2Y Receptor

Research Areas >> Metabolic Disease

EC50: 10 μM (P2Y2 receptor)[1]

Denufosol tetrasodium can be maintained above the EC50 point for 6 to 8 hours on the airway surface of airway cultures. Denufosol produces a significant increase in airway surface layer (ASL) height, reflecting P2Y2 receptor–mediated fluid secretion, which is terminated rapidly, likely reflecting P2Y2 receptor desensitization. The secreted ASL is reabsorbed over time, despite the continued presence of Denufosol at values exceeding its EC50, albeit at a rate slower than without Denufosol exposure (compared to vehicle group). This latter finding is consistent with the observation that P2Y2 receptor-mediated inhibition of Na+ absorption may persist for larger intervals than P2Y2 receptor activation of Cl- secretion[1]. Denufosol (INS37217) is a deoxycytidine-uridine dinucleotide with agonist activity at the P2Y2 receptor. In a concentration-dependent manner, Denufosol induces the mobilization of intracellular calcium in 1321N1 astrocytoma cells stably expressing human P2Y2 and P2Y4 receptors with EC50s of 0.22 and 0.8 μM, respectively. The efficacy of Denufosol is identical to that of UTP, indicating that Denufosol is a full agonist of P2Y2 and P2Y4 receptors. Denufosol is a weak agonist of P2Y6 receptors in which 100 μM Denufosol produces approximately 70% of the response observed with the natural agonist UDP[2].

The P2Y2 receptor agonist Denufosol (INS37217) improves the rate of retinal reattachment in animal models of induced retinal detachment (RD), and has been shown to also significantly enhance the rate of ERG recovery in a mouse model of RD. The identification of genes modulated by Denufosol may allow further drug discovery for treating RD and edema. RD is induced in Balb/cJ mice by transvitreal subretinal injections of 1 μL saline or of 1 μL of 10 μM Denufosol/saline to cause detachment and to expedite the rate of recovery[3].

Mice[3] Balb/cJ mice are used for microarray experiments. Subretinal injections are performed. Briefly, 1 μL saline or 1 μL saline/10 μM Denufosol is delivered to the subretinal space in Balb/cJ mice by transvitreal injection. This procedure creates a retinal hole that is self-sealing because material delivered will not diffuse into the vitreous. Mock injections consisting solely of a corneal puncture with no RD are also performed as a control. Mice are humanely killed at 2 hours, 24 hours, or 7 days after injection, and their retinas are harvested directly into reagent. For each time point, three separate pools of at least five retinas are collected for saline- and Denufosol -injected mice. After tissue homogenization, total RNA is isolated and is purified by a method for total isolation of RNA[3].

[1]. Button B, et al. Mechanosensitive ATP release maintains proper mucus hydration of airways. Sci Signal. 2013 Jun 11;6(279):ra46.

[2]. Yerxa BR, et al. Pharmacology of INS37217 [P(1)-(uridine 5')-P(4)- (2'-deoxycytidine 5')tetraphosphate, tetrasodium salt], a next-generation P2Y(2) receptor agonist for the treatment of cystic fibrosis. J Pharmacol Exp Ther. 2002 Sep;302(3):871-80.

[3]. Farjo R, et al. Expression profiling after retinal detachment and reattachment: a possible role for aquaporin-0. Invest Ophthalmol Vis Sci. 2008 Feb;49(2):511-21.