Increased exposure to blue or visible light, fluctuations in oxygen tension, and the excessive accumulation of toxic retinoid byproducts places a tremendous amount of stress on the retina. Reduction of visual chromophore biosynthesis may be an effective method to reduce the impact of these stressors and preserve retinal integrity. A class of non-retinoid, small molecule compounds that target key proteins of the visual cycle have been developed. The first candidate in this class of compounds, referred to as visual cycle modulators, is emixustat hydrochloride (emixustat). Here, we describe the effects of emixustat, an inhibitor of the visual cycle isomerase (RPE65), on visual cycle function and preservation of retinal integrity in animal models. Emixustat potently inhibited isomerase activity in vitro (IC50 = 4.4 nM) and was found to reduce the production of visual chromophore (11-cis retinal) in wild-type mice following a single oral dose (ED50 = 0.18 mg/kg). Measure of drug effect on the retina by electroretinography revealed a dose-dependent slowing of rod photoreceptor recovery (ED50 = 0.21 mg/kg) that was consistent with the pattern of visual chromophore reduction. In albino mice, emixustat was shown to be effective in preventing photoreceptor cell death caused by intense light exposure. Pre-treatment with a single dose of emixustat (0.3 mg/kg) provided a ~50% protective effect against light-induced photoreceptor cell loss, while higher doses (1–3 mg/kg) were nearly 100% effective. In Abca4-/- mice, an animal model of excessive lipofuscin and retinoid toxin (A2E) accumulation, chronic (3 month) emixustat treatment markedly reduced lipofuscin autofluorescence and reduced A2E levels by ~60% (ED50 = 0.47 mg/kg). Finally, in the retinopathy of prematurity rodent model, treatment with emixustat during the period of ischemia and reperfusion injury produced a ~30% reduction in retinal neovascularization (ED50 = 0.46mg/kg). These data demonstrate the ability of emixustat to modulate visual cycle activity and reduce pathology associated with various biochemical and environmental stressors in animal models. Other attributes of emixustat, such as oral bioavailability and target specificity make it an attractive candidate for clinical development in the treatment of retinal disease.

In the dark, photoreceptor outer segments contain high levels of cyclic guanosine 3 0-5 0 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 Mn 2þ-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.

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Vascular adhesion protein-1 (VAP-1) is an ectoenzyme that functions as a copper-containing amine oxidase and is involved in leukocyte adhesion at sites of inflammation. Inhibition of VAP-1 oxidative deamination has become an attractive target for anti-inflammatory therapy with demonstrated efficacy in rodent models of inflammation. A previous comparison of purified recombinant VAP-1 from mouse, rat, monkey, and human gene sequences predicted that rodent VAP-1 would have higher affinity for smaller hydrophilic substrates/inhibitors because of its narrower and more hydrophilic active site channel. An optimized in vitro oxidative deamination fluorescence assay with benzylamine (BA) was used to compare inhibition of five known inhibitors in recombinant mouse, rat, and human VAP-1. Human VAP-1 was more sensitive compared to rat or mouse VAP-1 (lowest IC50 concentration) to semicarbazide but was least sensitive to hydralazine and LJP-1207. Hydralazine had a lower IC50 in rats compared to humans, although not significant. However, the IC50 of hydralazine was significantly higher in the rat compared to mouse VAP-1. The larger hydrophobic compounds from Astellas (compound 35c) and Boehringer Ingelheim (PXS-4728A) were hypothesized to have higher binding affinity for human VAP-1 compared to rodent VAP-1 since the channel in human VAP-1 is larger and more hydrophobic than that in rodent VAP-1. Although the sensitivity of these two inhibitors was the lowest in the mouse enzyme, we found no significant differences between mouse, rat, and human VAP-1. Michaelis-Menten kinetics of the small primary amines phenylethylamine and tyramine were also compared to the common marker substrate BA demonstrating that BA had the highest affinity among the substrates. Rat VAP-1 had the highest affinity for all three substrates and mouse VAP-1 had intermediate affinity for BA and phenylethylamine, but tyramine was not a substrate for mouse VAP-1 under these assay conditions. These results suggest that comparing oxidative deamination in mouse and rat VAP-1 may be important if using these species for preclinical efficacy models.

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