Abstract
Purpose:
In the dark, photoreceptor outer segments contain high levels of cyclic guanosine 3′-5′ monophosphate (cGMP), which binds to ion channels, holding them open and allowing an influx of cations. Ion pumping activity, which balances cation influx, uses considerable amounts of adenosine triphosphate (ATP) and oxygen. Light reduces cation influx and thereby lowers metabolic demand. Blood vessels are compromised in the diabetic retina and may not be able to meet the higher metabolic demand in darkness. Emixustat is a visual cycle modulator (VCM) that reduces chromophore levels and, therefore, may mimic light conditions. We evaluated the effect of emixustat on oxygen consumption and cation influx in dark conditions.
Methods:
Cation influx was measured in rats using Mn2+-magnetic resonance imaging (MEMRI). Retinal oxygen profiles were recorded to evaluate oxygen consumption. In the MEMRI protocol, animals were treated with either emixustat or vehicle. In the oxygen protocol, animals were untreated or treated with emixustat.
Results:
In vehicle-treated animals, cation channel activity increased in the dark. Emixustat treatment reduced cation channel activity; activity was comparable to vehicle-treated controls in light conditions. In vehicle-treated animals, minimum retinal oxygen tension decreased as the retina recovered from a photobleach, indicating that more oxygen was being consumed. Emixustat treatment prevented the decrease in oxygen pressure after photobleach.
Conclusions:
Emixustat reduced the cation influx and retinal oxygen consumption associated with dark conditions. VCMs are a promising potential treatment for ischemic retinal neovascularization, such as that in diabetic retinopathy.
Photoreceptors are unlike most neurons because in the unstimulated state/dark state, photoreceptors are depolarized and, therefore, release the neurotransmitter (glutamate). In the dark, photoreceptor outer segments contain high levels of cyclic guanosine 3′-5′ monophosphate (cGMP), which binds to ion channels, holding them open and allowing an influx of cations (mostly Na
+ and Ca
2+). To complete the current loop, there is an efflux of K
+ from the inner segment . This influx of Na
+ and Ca
2+ ions and return efflux of K
+ is known as the dark current.
1 Large amounts of oxygen and adenosine triphosphate (ATP) are required to pump Na
+ back out and K
+ back in.
2 (Ca
2+ is restored by an outer segment Na
+/Ca
2+ exchanger, which adds to the Na
+ load on the cell
1.) Vessel damage associated with diabetic retinopathy (DR) reduces oxygen supply causing hypoxic conditions
3 that are exacerbated during dark adaptation.
4 Aberrant neovascularization, as in proliferative DR,
4 is probably a result of hypoxia.
5
Phototransduction is initiated when a photon of light activates rhodopsin. Rhodopsin consists of opsin protein covalently bound to the chromophore, 11-cis-retinal. When light strikes 11-cis-retinal, it is converted to all-trans-retinal and opsin undergoes a series of conformational changes resulting in a form called metarhodopsin II (Meta II), which activates an associated G protein, transducin, which leads to the activation of cGMP phosphodiesterase (PDE6), which breaks cGMP down into 5′-GMP. Reduced cGMP allows the ion channels to close, preventing cation influx, hyperpolarizing the cell, and stopping the release of glutamate. All-
trans-retinal is released from opsin creating apo-opsin. Apo-opsin can generate low level phototransduction (estimated activity 10
6-fold lower than that of Meta-II
6,7).
Emixustat (CAS number 1141777-14-1, ACU-4429) reduces chromophore levels
8 and, therefore, may mimic a state of constitutive phototransduction through the accumulation of apo-opsin, thereby decreasing the dark current.
9,10 Previously, we demonstrated that emixustat reduced neovascularization in the oxygen induced retinopathy (OIR) model.
11 In this study, we investigated the hypothesis that emixustat puts the photoreceptors in a state of partial light adaptation and reduced metabolism by testing the accumulation of ions using Mn
2+-magnetic resonance imaging (MEMRI) and measuring retinal oxygen profiles in rats treated with emixustat.
Emixustat depletes chromophore, which should generate apo-opsin. It was hypothesized that apo-opsin generated by emixustat treatment would initiate low level constitutive phototransduction. This would, in turn, reduce cGMP levels and ion channels would close, preventing cation influx, and reducing oxygen consumption. Our results supported this mechanism, showing that emixustat reduced cation influx during dark adaptation. These data indicated that emixustat can reduce the metabolically demanding dark current. We also demonstrated that PO2 remains higher in dark-adapted emixustat-treated rat retinas compared to untreated dark-adapted retinas, indicating that that less oxygen was consumed in the photoreceptors of emixustat treated retinas. Together, these data showed that emixustat can reduce metabolic requirements of dark conditions.
Decreasing the metabolic demand of dark with emixustat may be therapeutic for diseases such as DR. However, because emixustat inhibits the visual cycle, there is a practical concern as to how emixustat treatment would affect vision. Emixustat seems to have no effect on vision from cone photoreceptors, which are responsible for high acuity central vision. Emixustat has been administered in 11 completed clinical studies for up to 2 years. Photoreceptor activity can be assessed by ERG. In completed clinical studies where ERG data are available, emixustat suppressed recovery of the rod b-wave amplitude following bleaching light exposure in a dose-dependent and reversible manner. Cone ERGs were not significantly affected.
8,20 In addition, visual acuity under normal, photopic conditions was not affected. The most common adverse events, visual color distortions and delayed dark adaptation, exhibited a dose-dependent trend in incidence and are consistent with emixustat's mechanism of action, with preferential effects on rod function. Effects on color perception likely occurred through secondary rod-cone interactions.
This finding that emixustat affects the rod ERG, but not the cone ERG ,may be attributed to the greater light sensitivity of rods, compared to cones. However, there is compelling evidence that cones do not rely on RPE65 as the primary source of visual chromophore and, therefore, may not be significantly affected by specific inhibitors of RPE65.
21,22 These observations led to the belief that visual pigment is regenerated at a faster rate in cones compared to rods and/or that chromophore may be supplied more efficiently to cones than to rods, possibly with the involvement of an alternate intraretinal visual cycle.
Recent progress in the characterization of the intraretinal visual cycle has shown that emixustat did not compromise the ability of cones to maintain light sensitivity during exposure to bright light.
23 Only the late, RPE visual cycle–driven phase of cone dark adaptation was suppressed by emixustat. From this and other findings, the investigators concluded that while visual pigment regeneration mediated by RPE65 appears to contribute to regeneration of cone pigments under certain conditions, RPE65 is not essential to maintain light sensitivity of cones.
Previously, we showed that emixustat reduced retinal neovascularization in the oxygen induced retinopathy model.
11 It was hypothesized that emixustat could reduce neovascularization by reducing metabolic stress. Other investigators hypothesized that prevention of complete dark adaptation, via activation of rod phototransduction, may be effective in preventing hypoxia and preserving retinal vasculature.
4,24 Data shown here support the hypothesized mechanism of action for the reduction of neovascularization. Decreasing retinal oxygen demand in the setting of diseases, such as DR, may reduce hypoxia associated with these conditions, potentially leading to a therapeutic effect. Emixustat may be promising potential treatment for ischemic retinal neovascularization such as DR.
The authors thank Jeff Gregory, MD, for help reviewing the manuscript, Nathan Mata, PhD, for scientific conversations, and Jennifer C. Lau, PhD, for her contributions to the retinal PO2 measurements.
Disclosure: R. Kubota, Acucela (I, E, S), P; D.J. Calkins, Acucela (F); S.H. Henry, Acucela (I, E), P; R.A. Linsenmeier Acucela (F)