Investigation of the Mechanism of Action and IOP Lowering Ability of CB1 Agonists using Glauconix Biosciences’ 3D-HTM^TM Tissue Model

Abstract

Purpose : The potential of cannabinoids as a means to lower IOP has been well documented for several decades, but the mechanism of action is not fully understood. The psychoactive component of cannabis, Δ⁹-THC, has the ability to decrease IOP through the activation of CB1 cannabinoid receptors, making these receptors a potential therapeutic target for glaucoma. The presence of CB1 receptors in the trabecular meshwork and Schlemm’s canal highlights their potential involvement in IOP lowering through the conventional outflow tract. Using a glaucomatous 3D bioengineered human trabecular meshwork tissue (3D-HTM^TM) construct, the effect of CB1 receptor agonists on fibrotic markers and IOP modulation was studied.

Methods : 3D-HTM^TM constructs were cultured to confluence, treated with TGFb-2 (2.5 ng/mL) for 6 days to induce a glaucomatous state. Constructs were then exposed to two different CB1 receptor agonists, WIN-55,212-2 (10 μM) and Arvanil (10 μM) for 7 days. The expression of fibrotic markers, including α-smooth muscle actin, collagen IV and fibronectin, were assayed. The structural integrity of these markers was analyzed using confocal microscopy. In parallel, the responsiveness of these models to CB1 receptor agonists under flow was investigated by perfusion studies using a multichannel microfluidic system. The “outflow facility” of the tissue construct was determined by the ratio of Δ(flow rate)/Δ(pressure).

Results : Immunocytochemical analysis demonstrated significant structural changes of collagen IV and fibronectin expression across glaucomatous 3D-HTM^TM constructs after both WIN-55,212-2 and Arvanil treatments. Both CB1 agonists decreased collagen IV and fibronectin protein expression when compared to the glaucomatous constructs alone. Additionally, perfusion studies indicated that outflow facility increased with Arvanil treatment compared to glaucomatous constructs (0.71 + 0.3 μL/min/mm²/mmHg versus 0.40 + 0.09 μL/min/mm²/mmHg) (P>0.05).

Conclusions : We report that CB1 agonists induce changes in the ECM by decreasing collagen IV expression and thereby have potential anti-fibrotic effects in glaucomatous tissue. The decrease in collagen IV expression supports the increase in the outflow facility observed in CB1 receptor agonist Arvanil and thereby suggests a potential mechanism by which CB1 receptor agonists could be used as a glaucoma therapeutic.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.