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.
PnPP-19 was synthesized at Watsonbio (Houston, TX) and PnPP-19-FITC was synthesized at GenOne (Rio de Janeiro, RJ, Brazil). The peptide (4 mg) was dissolved in 1 mL of saline and 80 µg/eye (1.6 mM) were applied, as an eye drop (one drop was instilled). This dose was decided based on a previous study that evaluated the toxicity of PnPP-19 and its efficacy in healthy (normotensive) rats (unpublished data). One drop of Bimatoprost (Medley, Campinas, SP, Brazil) 0.3 mg/mL (equivalent to 8.8 µg/eye – 1 mM) was used. As vehicle, one drop of saline (0.9% NaCl) was used. The drop volume was the same for PnPP-19 and vehicle groups (i.e. 20 µL).
Evaluation of PnPP-19 Toxicity Through Hen's Egg Test in Chorioallantoic Membrane Test
IOP was evaluated, at the same time, in normotensive and hypertensive eyes by an experienced tonometrist, using a TonoPen Vet (Reichert, NY) that was calibrated before use. Non-sedated animals were carefully contained for the measurements. Four IOP readings were taken for each eye (with a standard error of < 5%), and their average was considered as the IOP value.
To confirm that PnPP-19 can permeate the cornea, three healthy rats received the peptide conjugated with FITC or vehicle in a lightless condition. After 2 hours, the IOP was checked and the animals were euthanized using an overdose of anesthetic with IP injection of 270/30 mg/kg of ketamine and xylazine, respectively. The eyes were removed and submitted to histological analysis. Images were obtained using a confocal microscope (LSM 880; Zeiss Jena, Aalen, Germany) with a 20× objective, scale bar = 50 µm. FITC was excited at 490 nm with emission at 526 nm. The FITC quantification of fluorescence was measured using the Image J software (National Institutes of Health, Bethesda, MD). At least 10 randomly selected fields of every section were used to assess relative fluorescence intensity, which was analyzed according to the number of pixels.
After ERG recordings or ocular hypertension induction and treatments, the animals were euthanized and eye tissues were processed for light microscopy. Eyes were enucleated and fixed in Davidson's solution (10% neutral phosphate-buffered formalin, 95% ethanol, glacial acetic acid, and ultrapure water 2:3:1:3, respectively). Samples were included in paraffin and 4-µm-thick sections were performed, stained with hematoxylin and eosin. The investigators taking the histological images masked to the treatment given to the eyes. Retinal ganglion cell (RGCs) were counted along a 200 µm linear region of the ganglion cell layer (GCL) either side of the optic nerve head (N = 6).
FITC-Conjugated PnPP-19 (PnPP-19-FITC) was Able to Permeate Cornea and Reduce IOP of Rats After Topical Administration
In the present work, we demonstrated that the synthetic peptide PnPP-19 is capable of reducing IOP in animals with healthy and hypertensive eyes, besides protecting against injury to the optic nerve, after topical administration as eye drops.
IOP is the critical risk factor for glaucoma, a neurodegenerative disease and frequent cause of blindness worldwide. POAG is associated with high IOP and is the leading cause of glaucomatous blindness, affecting 80 to 90% of patients with glaucoma. IOP reduction decreases the incidence rate and slows the progression of POAG.
9,30 Treatment of POAG requires pharmacotherapy as soon as ocular hypertension is detected. Despite the availability of several anti-glaucoma medications, there is still an important medical need in this field.
In this context, the peptide PnPP-19 emerges as an interesting therapeutic option because, besides reducing IOP, it has a short polypeptide chain without disulfide bridges, which eases its synthesis. All these features strongly indicate its viability for use and commercialization. In addition, we showed herein that this peptide is not toxic or irritating when applied to the CAM. In contrast, as expected, NaOH solution was severely irritating, as presented in the OII score table (
Table). This test is appropriate, because CAM is analogous to the human retina and its vasculature. Irritations at risk of vascular damage, such as bleeding, lysis, and coagulation, can be evaluated by HET-CAM test, which is optimal for the study of compound accuracy and safety for ocular application.
31,32
Distinct ERG components are related to different retinal structures, so ERG is often used to monitor the functional integrity of the retina.
33 ERG examination did not indicate any change by PnPP-19 even at high doses, suggesting safety and no internal retinal impairment (see
Fig. 2, Fig.
3). The histopathological analysis showed that peptide-treated cornea and retina have the same morphology compared to control (see
Fig. 4), indicating security for the use of the peptide in all concentrations tested.
The IOP of healthy rats decreased around 20% after 2 hours of instillation of a single dose of PnPP-19, and this effect lasted for 24 hours (
Fig. 5). This result highlights the effectiveness of this peptide, because if the desired IOP level is not achieved with a single dose or therapy, additional doses of medications may be included in the treatment regimen,
13 which may trigger several adverse symptoms. Therefore, topical administration, with a low frequency of treatment, is a valuable and interesting resource to have, and is also advantageous for individuals who have difficulties with medication adherence.
34
Recent evidence indicates that the NO signaling pathway plays an important role in ocular homeostasis, regulating AH drainage and, therefore, IOP. Contractile tissues form the anatomic ocular structures that regulate AH drainage. The TM cells are known to be highly contractile, analogous to vascular smooth muscle cells, in which the role of NO-cGMP signaling in endothelium-dependent relaxation is well understood.
15
Studies in animals have shown that NO-induced IOP reduction is mainly mediated by an increase in conventional outflow caused by a relaxation of the TM cells, and probably by Schlemm's canal permeability increase. The Schlemm’s channel cells rise endogenous NO generation in response to shear stress supporting the NO function in IOP homeostasis.
35,36 In addition, the therapeutic potential of NO has recently been validated in patients with POAG.
15
In the presence of oxygen, endogenous NO has a short half-life, which makes it difficult to be measured in biological systems. Intracellular NO can be easily oxidized to more stable compounds, such as nitrite (NO
2-) and nitrate (NO
3-).
37 For this reason, nitrite concentration measurement is a good method for indirectly inferring if there is any change in NO levels after drug administration.
Topical administration of PnPP-19 increased nitrite levels in the ocular tissue from the anterior chamber, which may simultaneously improve conventional AH flow. Moreover, FITC-labeled PnPP-19 assay indirectly revealed that this peptide can permeate corneal layers reaching tissues of the anterior chamber. It is possible that PnPP-19 improves NO release, evidenced by an increase in nitrite concentration, which promotes contractility changes that corroborate with IOP reduction, but more evidence is needed to prove this action.
Previous studies have shown that chronic administration of HA by intracameral injections induces significant IOP elevation and damage in the retina, which seems consistent with some characteristics of POAG,
12,38 in addition, it has been demonstrated that this model may be useful for pharmacological studies with drugs that affect the formation of aqueous humor or outflow or both,
12,39 being relevant to test the PnPP-19 action. Our results show that the IOP increased 32% after the intraocular injections of HA and the reduction in IOP triggered by PnPP-19 was also effective in the ocular hypertension model we used. This finding is therapeutically relevant given the clinical routine, highlighting that, despite reducing IOP by around 30% after 24 hours of a single instillation, PnPP-19 did not change MAP as previously described.
20,40
A commercial prostaglandin-analog antiglaucoma agent (Bimatoprost) was used to compare its effects with those of PnPP-19. IOP lowering remained for 24 hours after the treatment with a single topical instillation of PnPP-19. Bimatoprost, on the other hand, was not able to reduce IOP according to the established protocol (see
Fig. 8). It is possible that this lack of effect is associated with a difference in drug concentrations. However, Franca et al.
27 obtained good results using 9 µg of Bimatoprost for the treatment of glaucoma in rats, a similar dose as the one used in this work, but it was administered in an ocular insert.
When co-administered Bimatoprost and PnPP-19, the effect improved, leading to a higher reduction in IOP values, compared to Bimatoprost alone. Similar to the results observed in this study, latanoprostene bunod, an NO-donating prostaglandin F receptor agonist, and was more effective than the reference compound, latanoprost, in lowering IOP. In this case, the dual-mode of action, combining prostaglandin F receptor activation and NO donation, increased AH outflow through both unconventional and conventional pathways, simultaneously.
15 However, the interaction between PnPP-19 and Bimatoprost did not promote synergism in the effect, on the contrary, we observed that this interaction impaired the action of PnPP-19 because its effect was less if compared to the peptide alone. We believe that interaction between these drugs happened, and Bimatoprost may have impaired the binding of PnPP-19 to its target, and further investigation is needed to clarify this question.
Glaucoma is characterized as a slow degeneration of RGC followed by axon loss. It manifests as a progressive retinal nerve fiber layer thinning and optic nerve head cupping, which functionally results in a characteristic pattern of visual field loss.
13
Therefore, at the end of the treatment, histological sections of the retina and the optic nerve area were evaluated to verify the RGC loss and optic nerve head cupping. As shown, the vehicle-treated group showed significant RGC loss, whereas in the PnPP-19-treated group the RGC number was similar to that of healthy animals, suggesting that the treatment with PnPP-19 also promoted neuroprotection. However, future experiments are needed to confirm this last result.
The present work shows that an NO-inducer peptide, PnPP-19, when topically administered as eye drops, reduces IOP after a single dose, both in normotensive and ocular hypertensive rats, without eliciting apparent toxicity. Our results suggest that PnPP-19 is, probably, improving the conventional AH outflow and directly preventing the progression of optic nerve degeneration. Thus, this synthetic peptide has great potential to emerge as a promising drug for the treatment of glaucoma, isolated or in combination with conventional drugs.
Supported by fellowships and grants that were awarded by the Brazilian University UFMG (Universidade Federal de Minas Gerais), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), INCT - Nanotecnologia Farmacêutica (Instituto Nacional de Ciência e Tecnologia), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), BIOZEUS Biopharmaceutical S.A., and FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais).
A portion of this work was presented at the annual meeting of the North American Society of Toxinology - The Venom Week 2020, Gainesville, Florida, United States, Mar 2020.
Disclosure: C.N. da Silva, Biozeus (F); L.F.N. Dourado, Biozeus (F); M.E. de Lima, Biozeus (F); A. Silva Cunha-Jr, Biozeus (F)