NCX 667, a Novel Nitric Oxide Donor, Lowers Intraocular Pressure in Rabbits, Dogs, and Non-Human Primates and Enhances TGFβ2-Induced Outflow in HTM/HSC Constructs

Abstract

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.

Figure 1.

Synthetic pathway for NCX 667 ((S)-((3R,3aR,6R,6aR)-6-hydroxyhexahydrofuro[3,2-b]furan-3-yl) 5,6-bis(nitrooxy)hexanoate). Final yield of 10.7% (calculated on isomannide).

Figure 2.

Concentration-dependent changes in outflow facility in 3D-HTM/HSC constructs. The “outflow facility” of the bioengineered 3D-HTM/HSC model was calculated mathematically from the flow rate and pressure data collected during the perfusion study. For each concentration n = 4–6 individual perfusion studies were conducted, and the data are shown as mean ± SEM. Differences between samples were analyzed using one-way analysis of variance followed by Tukey post-tests.

Figure 3.

IOP-lowering effect of NCX 667 in ONT-rabbits. Intraocular pressures (A) and differences from vehicle (B) after instillation of NCX 667 (0.1%, N = 8; 0.3%, N = 8; 1%, N = 7) and the respective vehicle (phosphate buffer pH 6.0, Kolliphor EL 5%, DMSO 0.3%, BAC 0.02%, N = 5) were determined in ONT-rabbits. IOPs were determined using a pneumatonometer. Values reported at each time point result from two consecutive measurements taken one minute apart and averaged. The ΔΔIOP were calculated as follows: (Drug IOP_Tx − Drug IOP_T0) − (Veh IOP_Tx − Veh IOP_T0) where IOP_Tx and IOP_T0 are, respectively, the IOP at the time of interest and at baseline. Data are reported as mean ± SEM. ^*P < 0.05 versus the respective vehicle group, t-test multiple comparisons.

Figure 4.

IOP-lowering effect in ONT-dogs. Intraocular pressure (A, C) and changes from vehicle and baseline (B, D) after instillation of NCX 667 (0.1%, and 1%) and the respective vehicle (phosphate buffer pH 6.0, Kolliphor EL 5%, DMSO 0.3%, BAC 0.02%) were determined in ONT-dogs (N = 8, four females and four males). ΔΔIOPs were calculated as follows: (Drug IOP_Tx − Drug IOP_T0) − (Veh IOP_Tx − Veh IOP_T0) where IOP_Tx and IOP_T0 are, respectively, the IOP at the time of interest and at baseline. Data are reported as mean ± SEM. ^*P < 0.05 versus the respective vehicle group, t-test multiple comparisons.

Figure 5.

Changes in cGMP in ocular tissues after NCX 677 topical dosing in ONT-rabbits. The cGMP levels after topical dosing of NCX 667 (1%) were monitored in AH (A) or iris/ciliary body and retina (B) of ONT-rabbits at the indicated time points. Data are reported as mean ± SEM, n = 3 eyes/time point. ^*P < 0.05 versus respective vehicle; two-way analysis of variance followed by Dunnett's multiple comparisons test.