PnPP-19 Peptide as a Novel Drug Candidate for Topical Glaucoma Therapy Through Nitric Oxide Release

Abstract

Purpose: Evaluation of PnPP-19 safety and efficacy in reducing the intraocular pressure (IOP) of animals with healthy (normotensive) and ocular hypertensive eyes. PnPP-19 is a synthetic peptide designed from Phoneutria nigriventer spider toxin PnTx2-6.

Methods: Toxicity tests used chicken chorioallantoic membranes. Electroretinograms (ERGs) were recorded before and after administration of different doses of PnPP-19 on the eyes of Wistar rats. Histological sections of corneas and retinas were prepared. The efficacy of PnPP-19 in reducing IOP was evaluated for normotensive and ocular hypertensive animals using a tonometer. Ocular hypertension was induced in the right eye through injection of hyaluronic acid (HA) into the anterior chamber. ERG was recorded before and after glaucoma induction. The eyes were enucleated, and the corneas and retinas were histologically evaluated.

Results: PnPP-19 showed no toxicity, being safe for ocular application. A single topical instillation of one eye drop of the peptide solution was able to reduce IOP, both in healthy and ocular hypertensive rats, for 24 hours, without eliciting any apparent toxicity. PnPP-19 is a nitric oxide inducer and the results suggest that it may improve the conventional outflow of aqueous humor (AH), preventing the progression of optic nerve degeneration.

Conclusions: PnPP-19 has great potential to emerge as a promising drug for the treatment of ocular hypertension.

Translational relevance: We regard our findings as exciting progress in translational glaucoma research, combining drug discovery, natural product research, and pharmacology, which may contribute to the establishment of new therapies for the treatment of this disease.

Keywords: glaucoma; glaucoma medications; nitric oxide.

Figure 1.

Microscopy analysis of the effect of PnPP19 on CAM vessels. Images of HET-CAM after 300 seconds of exposure to: (A) 0.1M NaOH (positive control); (B) 0.9% NaCl (negative control) and treatment with PnPP-19 eye drops at the following concentrations: (C) 40 µg; (D) 80 µg; (E) 160 µg, and (F) 320 µg.

Figure 2.

ERG curve in scotopic conditions. The amplitude and implicit time values were analyzed (A) 0.01 and (B) 3.0 cd.s/m²) after: 1, 7, and 15 days of exposure to PnPP-19 (40, 80, and 160 µg). As a control, we used the eyes before the administration of the peptide solution (N = 10).

Figure 3.

Mean ± standard derivation of the amplitude and implicit time. The amplitude (microvolts - µV) and implicit time (millisecond - ms) values of a- and b-waves in scotopic conditions (0.01 and 3.0 cd.s/m²) after PnPP-19 treatment. As a control we used the eyes before exposing to the peptide. ERG curve patterns were analyzed using Shapiro-Wilk test followed by Kruskal-Wallis and post-test of Dunn (N = 10).

Figure 4.

PnPP-19 does not alter cornea and retina morphology. Sequence of illustrative photographs of histological layers of the cornea (A) and retina (B) after PnPP-19 instillation in the doses of 40, 80, and 160 µg and Control (N = 6). Cornea layers: epithelium (Epit), stroma, endothelium (Endo). Retina layers: ganglion cell layer (Gcl), inner nuclear layer (Inl), and outer nuclear layer (Onl). Digital images were obtained with a 20× objective using a microscope (Apotome.2, Zeiss, Germany).

Figure 5.

PnPP-19 (80 µg/eye) reduces IOP in normotensive (healthy) rats. Comparison between PnPP-19 and Vehicle. Results are expressed in mm Hg (N = 6). Asterisks represent statistical difference compared to control * P < 0.5; ** P < 0.01; *** P < 0.001, 2-way ANOVA with Bonferroni post-test.

Figure 6.

Permeation of PnPP-19 through the cornea. Fluorescence promoted by PnPP-19-FITC showed that this peptide was able to permeate through the cornea (N = 3). Asterisks represent statistical difference compared to vehicle *** P <0.001, 2-way ANOVA with Bonferroni post-test.

Figure 7.

Effect of PnPP-19 instillation on nitrite concentration (NO^2-) in the homogenized eye tissues. The tissues of the normotensive (healthy) eyes were collected 2 hours after local instillation of vehicle (saline) or PnPP-19 (80 µg/eye). Each column represents the mean ± SEM (N = 6). *** P < 0.001, the Student t test for nonpaired data.

Figure 8.

PnPP-19 reduces IOP in rats with ocular hypertension. Comparison between treated hypertensive eyes with Vehicle (saline – NaCl 0.9%), PnPP-19 (80 µg/eye), Bimatoprost (8.8 µg/eye), PnPP-19 + Bimatoprost (PnPP-19 at 80 µg/eye plus Bimatoprost at 8.8 µg/eye) and healthy eyes. Results expressed in mm Hg (N = 6). Asterisks represent statistical difference in relation to the untreated * P < 0.5; ** P < 0.01; *** P < 0.001, 2-way ANOVA with Bonferroni post-test.

Figure 9.

Histologic analysis of healthy and ocular hypertension rats. The vehicle-treated ocular hypertension animals exhibited an increase in the excavation of the optic nerve (red arrows) and increase in the number of cytoplasmic vacuolization in the GCL (black arrows) (B, D) if compared to healthy rats (A,C). Besides, the INL displays more edema, pyknotic nuclei, and cellular disorganization (black arrows) (D). The ONL exhibits a decreased cell number, and greater edema and cell disorganization compared with the healthy retina D. Treatment with PnPP-19 (E,G) reduced histologic damage if compared to vehicle-treated glaucoma B and D or Bimatoprost (F,H). Healthy A and C; ocular hypertension vehicle-treated B and D; ocular hypertension treated with PnPP-19 E and G Bimatoprost F and H. Retina layers: ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL). Digital images were obtained using a microscope (Apotome.2, Zeiss, Germany) with a 10× and 20× objective, scale bar = 50 µm.

Figure 10.

Increases in the IOP were accompanied by reductions in the number of RGCs (retinal ganglion cells). There was a smaller number of RGCs in the retinas of ocular hypertension animals compared to healthy rats. Ocular hypertension animals treated with PnPP-19 have more RGCs than vehicle treated ocular hypertension rats, and were not statistically different compared to healthy rats (N = 6). Asterisks represent statistical difference concerning the untreated * P < 0.05; ** P < 0.01, 2-way ANOVA with Bonferroni post-test.