While clamping pressure at 15 mm Hg, outflow of both netarsudil-M1–treated and control groups (
n = 5 each) was continually measured and outflow facility was calculated at baseline and at 30 minutes, 2 hours, and 3 hours after drug or vehicle application (
Fig. 4A;
Table). Outflow facility increased over time in the netarsudil-M1–treated group, while remaining unchanged in the control group. Outflow facility of the netarsudil-M1 group versus the GPBS group was 0.28 ± 0.05 vs. 0.21 ± 0.03 μL/min/mm Hg (
P = 0.08; 1-tailed, paired
t-test) at baseline, 0.34 ± 0.05 vs. 0.20 ± 0.03 μL/min/mm Hg (
P = 0.005) at 30 minutes after perfusion, 0.40 ± 0.04 vs. 0.21 ± 0.03 μL/min/mm Hg (
P = 0.0002) at 2 hours after perfusion, and 0.43 ± 0.05 vs. 0.21 ± 0.04 μL/min/mm Hg (
P = 0.0002) at 3 hours after perfusion. When compared to its own baseline, the netarsudil-M1 group had shown a significant increase after 30 minutes perfusion (
P = 0.046 at 30 minutes, 0.004 at 2 hours, and 0.0006 at 3 hours; 1-tailed, paired
t-test). In contrast, the GPBS group had no change in outflow facility over the entire 3-hour perfusion when compared to its own baseline (
P = 0.35 at 30 minutes, 0.42 at 2 hours, and 0.45 at 3 hours).
Percentage change in outflow facility in netarsudil-M1–treated eyes increased in a perfusion time–dependent manner, while remaining unchanged in control eyes (
Fig. 4B). The percentage change in outflow facility of the netarsudil-M1 versus GPBS groups was 24.66% ± 12.57% vs. −2.21% ± 5.62% (
P = 0.08; 1-tailed, paired
t-test) at 30 minutes, 50.13% ± 14.64% vs. −3.35% ± 7.24% (
P = 0.02) at 2 hours, and 59.70% ± 13.65% vs. −1.70% ± 10.34% (
P = 0.02) at 3 hours.
Outflow facility change from baseline at the last time point (3 hours) was further analyzed by using the linear mixed-effects model for repeated measures. The analysis included the fixed categorical effects of treatment, time point, treatment-by-time-point interaction, and the covariate of baseline outflow facility measurement, with the baseline outflow facility measurement-by-time-point interaction adjusted in the model. The restricted maximum likelihood method with a compound symmetry covariance structure shared across treatment groups was used to model the within-patient errors. The Kenward-Roger method was used to estimate denominator degrees of freedom and adjust standard errors. The least square (LS) means of the control and treatment group were −0.02 and 0.12, respectively. At the 3-hour time point, the difference in LS mean change from baseline between the control and treatment groups (−0.14) was statistically significant (P < 0.001, t = −9.47 and df = 18.5). This increasing value in outflow facility change from baseline at the last time point (3 hour) indicates a positive drug effect.