Discussion
The most important facet of JV-GL1 pharmacology is arguably its ultra-long duration of action (Woodward et al ., 2019a). The present studies have demonstrated that this prolonged effect on intraocular pressure is entirely mediated by a single pharmacological entity, the EP2 receptor. Class selective long-acting β2-agonists (LABAs) and muscarinic antagonists (LAMAs) have previously been discovered (Wold et al., 2019; Wendell et al., 2020) but their clinical dosing regimen is once daily, even when used in combination (Maqsood et al., 2019). For JV-GL1, a once weekly dosing regimen is feasible. Further research on JV-GL1 and the EP2 receptor may lead to a wide range of small molecules dosed once-weekly that offer improved disease control and lower drug costs. At this juncture, the existing data suggests JV-GL1 may be an important new drug for women’s health and inflammation (Coleman et al ., 2019). For glaucoma, the data indicate that JV-GL1 is a realistic proposition for a breakthrough drug in treating glaucoma (Colemanet al ., 2018; Woodward et al., 2019a).
The long-acting effects of JV-GL1 on IOP in monkeys extend far beyond time points when detectable levels of the drug can be measured in anterior segment tissues of the eye (Woodward, et al. , 2019a). JV-GL1 was designed as an EP2 receptor agonist and the long acting effects of a week or more were quite unexpected, given that the ocular hypotensive duration of action of the numerous EP2 agonist family is essentially one day (Woodward,et al. , 2019b). This raised the possibility that the effects of JV-GL1 on IOP occurred as a result of two separate pharmacological activities; an initial EP2 receptor mediated effect followed by a secondary long-acting phase that was independent of EP2 receptors. In order to address this question, we studied the effects of JV-GL1 in gene deleted mice (Saeki et al. , 2009). Our studies demonstrate that the entire duration of ocular hypotensive action of JV-GL1 is EP2 receptor mediated.
A single topical application of 0.01% JV-GL1 significantly reduced IOP in both ocular normotensive and steroid-induced ocular hypertensive mice after 3 hours. However, only steroid treated mice exhibited a long-term pressure reduction, persisting for 4 to 6 days. JV-GL1 was incapable of reducing IOP in mice lacking the EP2 receptor under both ocular normotensive and steroid ocular hypertensive conditions. Conventional interpretation of G-protein coupled receptor signalling would suggest that upon ligand binding to the EP2receptor Gαs coupling activates adenylate cyclase which converts adenosine triphosphate to the second messenger, cyclic adenosine monophosphate (cAMP) (Regan et al. , 1994; Pierceet al. , 1995). The cAMP-dependent pathway promotes matrix metalloproteinase (MMP’s) secretion (Shim, Kim and Ju, 2017). MMP’s break down extracellular matrix material in the outflow tissues of the eye, reducing resistance and lowering IOP (Nilsson et al. , 2006).
In this study, topical JV-GL1 did not significantly affect outflow facility in the steroid ocular hypertensive mouse eye, nor in ocular normotensive mouse eyes perfused with de-esterified JV-GL1 over an acute time-scale. A study using cellular dielectric spectroscopy found JV-GL1 to have high activity in human ciliary muscle cells but little activity in human trabecular meshwork cells (3.9 nM vs >10 µM, EC50 values respectively) (Woodward, et al. , 2019a). This suggests a uveoscleral mechanism of action for JV-GL1, which correlates with the minimal effects on outflow facility observed in this study and with the mechanism of action proposed for the standard and most studied EP2 agonist, butaprost (Nilssonet al. , 2006). Our perfusion results largely corroborate those obtained from perfusion studies in monkeys, where JV-GL1 did not significantly affect outflow facility. However, in monkeys JV-GL1 significantly increases uveoscleral outflow, as measured by fluorophotometry, and significantly reduces inflow by 20% over an acute timescale (Woodward, et al. , 2019a). A limitation of measuring outflow facility in enucleated mouse eyes is that inflow is essentially terminated once the eye is removed and the pressure independent nature of the uveoscleral pathway makes it challenging to measure using a pressure controlled perfusion system. Thus, any effect JV-GL1 may have on inflow or uveoscleral outflow cannot be measured. Unfortunately in monkey studies, inflow measurements were not continued over the time-course of IOP measurements, and the relationship between a reduction in inflow and IOP suppression remains unanswered.
In monkeys, JV-GL1 exhibited long-term IOP effects in both ocular normotensive and laser-induced ocular hypertensive animals, and demonstrated considerably greater pressure reduction (Woodward et al. , 2019a) compared to the observations made in this study with mice. These inconsistencies may simply be due to species differences. The relative size of the ciliary muscle is much smaller in mice than in primates (Ko and Tan, 2013) as mice do not accommodate for vision (Tamm and Lutjen-Drecoll, 1996). If JV-GL1 is thought to work primarily via the uveoscleral route, then the more developed ciliary muscle of the monkey, which is in frequent use and therefore containing greater amounts of extracellular matrix material, would be expected to respond better to remodelling effects of MMP’s than the more vestigial ciliary muscle of the mouse.
Alternatively, it may be the different methods employed to induce ocular hypertension. Unlike laser-induced ocular hypertension, the steroid induced ocular hypertensive model relies on the persistent presence of dexamethasone in the eye, eluting from periocular depots of dexamethasone loaded nanoparticles. Steroid ocular hypertension is brought about by dexamethasone induced changes in extracellular matrix deposition and stiffness in the trabecular meshwork (Overby et al. , 2014; Raghunathan et al. , 2015). Increased extracellular matrix deposition is likely due to dexamethasone inhibiting MMP’s, either through inhibition of transcription factors (Forster et al. , 2007) or by inducing tissue inhibitors of matrix metalloproteases (MMP’s) (Xu et al. , 2001). Dexamethasone may have similar effects on the uveoscleral pathway. MMP’s 2, 3 & 9 were found to be significantly reduced in ciliary body explants treated with dexamethasone, with MMP-3 eliminated after 72 hours of dexamethasone treatment (el-Shabrawi et al. , 2000). MMP-3 can break down a diverse range of extracellular matrix components (Matrisian, 1990). What is more, dexamethasone has been shown to inhibit the production of endogenous prostaglandins in human trabecular meshwork cells and scleral fibroblasts (Gerritsen et al. , 1986).
Prostaglandins have been shown to induce MMP production in the ciliary body (el-Shabrawi et al. , 2000). These MMP’s are thought to be released into the aqueous and diffuse into the ciliary muscle where they break down extracellular matrix components between the longitudinal fibres of the ciliary muscle, allowing less restricted flow into the choroid (Ocklind, 1998). Consequently, we can speculate that JV-GL1 in the steroid ocular hypertensive mouse model would have to compete against the pro-fibrotic effects of constant dexamethasone delivery to the eye, limiting its ability to lower IOP to the same degree observed in the laser-induced ocular hypertensive monkey model, where no dexamethasone is present.
It is of interest that JV-GL1 has an effect on inflow in monkeys, as this has not been demonstrated with any other EP2agonists to date (Woodward, et al. , 2019b). The most studied EP2 agonist, butaprost, is efficacious at lowering IOP but has a short duration of action, with no effect on inflow and no effect on conventional outflow facility. The entirety of butaprost effects appear to be mediated by changes in uveoscleral outflow, specifically ciliary muscle resistance (Nilsson et al. , 2006). Therefore, JV-GL1 behaves somewhat differently when compared to other EP2 agonists. Aqueous flow can be reduced by direct activators of adenylate cyclase such as forskolin (Caprioli et al. , 1984) and cholera toxin (Gregory et al. , 1981), via a cAMP-dependent process. In the latter case, reduction in IOP persisted for 6 days after a single intravitreal injection.
It is, of course, highly unlikely that JV-GL1 is directly and/or irreversibly activating adenylate cyclase. However, recent research has disrupted the conventional view of G-protein coupled receptor signalling, whereby active receptor conformations engage and activate only one of four classes of cytoplasmic heterotrimeric G-proteins (Gαi/o,s, Gαq/11 and Gα12) and a more promiscuous model, whereby receptors can bind to more than one G-protein subtype, eliciting multiple G-protein dependent signalling events has emerged (Stallaert, et al., 2011; Hermans, 2003). Recent work investigating EP2receptor activation with butaprost, demonstrated not only the conventional Gαs – cAMP pathway activation but also Gαq/11 – calcium pathway activation upon ligand binding (Kandola et al. , 2014). Therefore, like forskolin and cholera toxin, which directly activate adenylate cyclase and thereby stimulate only the cAMP pathway, JV-GL1 may induce signal bias (Wold et al., 2019) and signal primarily via the Gαs-cAMP pathway by virtue of a novel interaction with the EP2 receptor. A study using cellular dielectric spectroscopy demonstrated that the EP2 antagonist AH6809 could not completely block JV-GL1 at the EP2 receptor, in human ciliary muscle cells (Woodward, et al. , 2019a). This finding suggests that JV-GL1 may be binding to a possible allosteric site on the EP2receptor where endogenous PGE2 does not bind, or bind to the EP2 receptor in a different way. Allosteric binding and a potential Gαs-cAMP pathway bias may be responsible for JV-GL1 long duration effects. This would set it apart from unbiased EP2 agonists such as butaprost and merit further research into its mechanism of action.
The long duration of action of JV-GL1 may result from allosteric binding, thereby implying that the binding of other anti-glaucoma EP2 agonists (Woodward et al., 2019b) to their target receptor is orthosteric. The long duration of action on IOP and the inherent activity on human ciliary smooth muscle cells could be indicative of JV-GL1 as a positive allosteric modulator. One, and only one, possible published explanation (Jiang et al., 2010) directly relevant to the ultra-long action of JV-GL1 on IOP involves allosteric modulation of the EP2 receptor. More pertinent to the extended ocular hypotensive activity is that allosteric potentiation of EP2 receptor activity is favoured by an ester moiety compared to a carboxylate22. The unexpected activity of JV-GL1 on ciliary smooth muscle cells correlates with manifestation of activity in intact cells rather than cell-free assays for EP2 allosteric potentiators22. JV-GL1 would be an ideal compound for elucidating the crystal structure of the EP2 and thereby gain valuable insights into orthosteric and allosteric binding sites and drug design.
In summary, the long-acting effects of JV-GL1 on IOP are EP2 receptor mediated and this provides an intriguing avenue of future investigation with respect to mechanisms that impart such extended biological activities. Beyond the significant potential benefits of JV-GL1 for improved glaucoma treatment, there are also implications for pre-term labour, asthma, and the anti-inflammatory effects of EP2 agonists. Future research may reveal similar advantages for compounds designed to be ultra-long acting at other GPCRs.