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Purpose: Determine whether NCX 470, a nitric oxide (NO)-donating bimatoprost with clinically demonstrated intraocular pressure (IOP)-lowering effects, improves ocular hemodynamics and retinal physiology. Methods: Endothelin-1 (ET-1)-induced ischemia/reperfusion model in New Zealand white rabbits was used. ET-1 was injected next to the optic nerve twice/week (Monday and Thursday) for 6 weeks. Starting on week 3, animals received NCX 470 (0.1% bid, 6 days/week Monday-Saturday) or vehicle until the end of ET-1 treatment. IOP, ophthalmic artery resistive index (OA-RI) and retina physiology (electroretinogram, ERG) were determined before dosing and at different times post-dosing. All measurements were taken on Mondays before the AM daily dosing (36 h treatment-free). Finally, oxidative stress markers were determined in dissected retina and iris/ciliary body of treated eyes. Results: Injection of ET-1 progressively increased IOP (20.7 ± 0.6, 24.9 ± 1.2, and 27.0 ± 0.6 mmHg at baseline, week 2 and 6, respectively) and OA-RI (0.30 ± 0.02, 0.39 ± 0.02, and 0.42 ± 0.03 at baseline, week 2 and 6, respectively) and reduced rods and/or cones response as indicated by changes in ERG amplitudes under different stimulating conditions. NCX 470 re-established baseline IOP (21.8 ± 1.0 mmHg), OA-RI (0.33 ± 0.02), and ERG amplitude by week 6 (mostly rod response, 0.01 Dark_A Veh_6week  = 32.2 ± 3.0 μV and 0.01 Dark_A NCX470_6week 44.3 ± 4.5 μV; mostly cone response, 3.0 Dark_A Veh_6week  = 87.6 ± 10.1 μV and 3.0 Dark_A NCX470_6week  = 122.8 ± 11.4 μV; combined rod/cone response, 3.0 Light_A Veh_6week  = 49.8 ± 6.5 μV and 3.0 Light_A NCX470_6week  = 64.2 ± 6.8 μV). NCX 470 also reversed ET-1-induced changes in glutathione and manganese superoxide dismutase (oxidative stress markers) in retina and iris/ciliary body. Conclusions: Repeated ocular topical dosing with NCX 470 reverses ET-1-induced changes in IOP, OA-RI, and ERG suggesting improved ocular hemodynamics and retinal physiology likely independently from its demonstrated IOP-lowering effect.

Purpose: NCX 667, a novel nitric oxide (NO) donor with an isomannide core, was characterized for its IOP-lowering ability in animal models of ocular hypertension and glaucoma. Bioengineered human trabecular meshwork/Schlemm's canal (HTM/HSC) constructs were used to explore the mode of action., Methods: Ocular normotensive New Zealand white (NZW) rabbits (ONT-rabbits), spontaneously ocular hypertensive pigmented Dutch-belted rabbits (sOHT-rabbits), hypertonic saline (5%)-induced transient ocular hypertensive NZW rabbits (tOHT-rabbits), ocular normotensive Beagle dogs (ONT-dogs), and laser-induced ocular hypertensive cynomolgus monkeys (OHT-monkeys) were used. NCX 667 or vehicle (30 µL) was instilled in a crossover, masked fashion and intraocular pressure (IOP) measured before dosing (baseline) and for several hours thereafter. The ONT-rabbits were used for cyclic guanosine monophosphate (cGMP) determination in ocular tissues after ocular dosing with NCX 667. Transforming growth factor-beta2 (TGFβ2) (2.5 ng/mL, six days)-treated HTM/HSC constructs were used to address changes in outflow facility., Results: NCX 667 resulted in robust and dose-dependent IOP decrease in all models used. Maximal IOP-lowering efficacy at 1% was -4.1 ± 0.6, -12.2 ± 2.7, -10.5 ± 2.0, -5.3 ± 0.8, and -6.6 ± 1.9 mmHg, respectively, in ONT-dogs, sOHT-rabbits, tOHT-rabbits, ONT-rabbits, and OHT-monkeys. In ONT-rabbits NCX 667 (1%) increased cGMP in aqueous humor (AH) but not in retina and iris/ciliary body. NCX 667 concentration-dependently increased outflow facility in TGFβ2-treated HTM/HSC constructs (outflow facility, 0.10 ± 0.06 and 0.30 ± 0.10 µL/min/mmHg/mm2, respectively, in vehicle- and NCX 667-treated constructs)., Conclusions: NCX 667 leads to robust IOP lowering in several animal models. Evidence in HTM/HSC constructs indicate that the IOP reduction likely results from NO-mediated increase of the conventional outflow pathway. Other mechanisms including changes in AH production and episcleral vein pressure may not be excluded at this time.

Intraocular pressure (IOP) has a tendency to fluctuate throughout the day, reaching its peak in the early morning in healthy subjects or glaucoma patients. Likewise, histamine tone also fluctuates over time, being lower at nighttime. Numerous studies have demonstrated a correlation between short-term IOP fluctuation and glaucoma progression; however, it has not yet been determined whether histamine plays a role in IOP fluctuations. The aim of this research was to establish the distribution of the histamine receptor proteins and respective mRNAs in the eye by western blot, immunohistochemistry and RT-PCR in New Zealand rabbits. Furthermore, we used a transient ocular hypertension (OHT) model induced by injection of 50 µL of 5% hypertonic saline into the vitreous and a stable OHT model (100 µL 0.1% carbomer in the anterior chamber) to address the potential IOP-lowering ability of H₃ receptor (H₃R) antagonists (ciproxifan, DL76 and GSK189254). IOPs were performed with a Tono-Pen at baseline and 60, 120 and 240 min post treatment after transient OHT induction and, every day for 12 days in the stable OHT model. All histamine receptor subtypes were localized in the rabbit retina and ciliary body/trabecular meshwork. All the treatments lowered IOP in a dose-dependent fashion between 0.3% and 1%. More specifically, the effects were maximal with ciproxifan at 60 min post-dose (IOP 60 change = -18.84 ± 4.85 mmHg, at 1%), remained stable until 120 min (IOP 120 change = -16.38 ± 3.8 mmHg, at 1%) and decayed thereafter to reach baseline values at 240 min. These effects were highly specific and dependent on histamine release as pre-treatment with imetit (H₃R agonist, 1%) or pyrilamine (H₁R antagonist, 1%) largely blocked ciproxifan-mediated effects. Color Doppler ultrasound examination was performed to evaluate changes in ophtalmic artery resistivity index (RI) before and after repeated dosing with DL 76, GSK189254, ciproxifan and timolol. Chronic treatments with H₃R antagonists and timolol improved the vascular performance of ophthalmic arteries and reduced retinal ganglion cell death. Oxidative stress was also reduced and measured 8-Hydroxy-2'-deoxyguanosine (8OH d G) expression, and by dihidroethydium (DHE) staining. These results demonstrated that the histamine system participates in IOP regulation and that H₃R antagonists could represent a future promising therapy for glaucoma. Further studies should be focused on the long-term IOP circadian fluctuations.