Neuroprotective and Anti-Inflammatory Activities of Hybrid Small-Molecule SA-10 in Ischemia/Reperfusion-Induced Retinal Neuronal Injury Models.

Embolism, hyperglycemia, high intraocular pressure-induced increased reactive oxygen species (ROS) production, and microglial activation result in endothelial/retinal ganglion cell death. Here, we conducted in vitro and in vivo ischemia/reperfusion (I/R) efficacy studies of a hybrid antioxidant-nitric oxide donor small molecule, SA-10, to assess its therapeutic potential for ocular stroke. METHODS: To induce I/R injury and inflammation, we subjected R28 and primary microglial cells to oxygen glucose deprivation (OGD) for 6 h in vitro or treated these cells with a cocktail of TNF-α, IL-1ß and IFN-γ for 1 h, followed by the addition of SA-10 (10 µM). Inhibition of microglial activation, ROS scavenging, cytoprotective and anti-inflammatory activities were measured. In vivo I/R-injured mouse retinas were treated with either PBS or SA-10 (2%) intravitreally, and pattern electroretinogram (ERG), spectral-domain optical coherence tomography, flash ERG and retinal immunocytochemistry were performed. RESULTS: SA-10 significantly inhibited microglial activation and inflammation in vitro. Compared to the control, the compound SA-10 significantly attenuated cell death in both microglia (43% vs. 13%) and R28 cells (52% vs. 17%), decreased ROS (38% vs. 68%) production in retinal microglia cells, preserved neural retinal function and increased SOD1 in mouse eyes. CONCLUSION: SA-10 is protective to retinal neurons by decreasing oxidative stress and inflammatory cytokines.

Therapeutic Potential of Antioxidants and Hybrid TEMPOL Derivatives in Ocular Neurodegenerative Diseases: A Glimpse into the Future.

Reactive oxygen species play a significant role in the pathogenesis of various ocular neurodegenerative diseases especially glaucoma, age-related macular degeneration (AMD), and ocular ischemic stroke. Increased oxidative stress and the accumulation of ROS have been implicated in the progression of these diseases. As a result, there has been growing interest in exploring potential therapeutic and prophylactic strategies involving exogenous antioxidants. In recent years, there have been significant advancements in the development of synthetic therapeutic antioxidants for targeting reactive oxygen species (ROS) in neurodegenerative diseases. One area of focus has been the development of hybrid TEMPOL derivatives. In the context of ocular diseases, the application of next-generation hybrid TEMPOL antioxidants may offer new avenues for neuroprotection. By targeting ROS and reducing oxidative stress in the retina and optic nerve, these compounds have the potential to preserve retinal ganglion cells and trabecular meshwork and protect against optic nerve damage, mitigating irreversible blindness associated with these diseases. This review seeks to highlight the potential impact of hybrid TEMPOL antioxidants and their derivatives on ocular neurodegenerative disorders.

Modulation of Mitochondrial Metabolic Parameters and Antioxidant Enzymes in Healthy and Glaucomatous Trabecular Meshwork Cells with Hybrid Small Molecule SA-2.

Oxidative stress (OS)-induced mitochondrial damage is a risk factor for primary open-angle glaucoma (POAG). Mitochondria-targeted novel antioxidant therapies could unearth promising drug candidates for the management of POAG. Previously, our dual-acting hybrid molecule SA-2 with nitric oxide-donating and antioxidant activity reduced intraocular pressure and improved aqueous humor outflow in rodent eyes. Here, we examined the mechanistic role of SA-2 in trabecular meshwork (TM) cells in vitro and measured the activity of intracellular antioxidant enzymes during OS. Primary human TM cells isolated from normal (hNTM) or glaucomatous (hGTM) post-mortem donors and transformed glaucomatous TM cells (GTM-3) were used for in vitro assays. We examined the effect of SA-2 on oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in vitro using Seahorse Analyzer with or without the oxidant, tert-butyl hydroperoxide (TBHP) treatment. Concentrations of total antioxidant enzymes, catalase (CAT), malondialdehyde (MDA), and glutathione peroxidase (GPx) were measured. We observed significant protection of both hNTM and hGTM cells from TBHP-induced cell death by SA-2. Antioxidant enzymes were elevated in SA-2-treated cells compared to TBHP-treated cells. In addition, SA-2 demonstrated an increase in mitochondrial metabolic parameters. Altogether, SA-2 protected both normal and glaucomatous TM cells from OS via increasing mitochondrial energy parameters and the activity of antioxidant enzymes.

Therapeutic applications of CRISPR/Cas9 gene editing technology for the treatment of ocular diseases.

Ocular diseases are a highly heterogeneous group of phenotypes, caused by a spectrum of genetic variants and environmental factors that exhibit diverse clinical symptoms. As a result of its anatomical location, structure and immune privilege, the eye is an ideal system to assess and validate novel genetic therapies. Advances in genome editing have revolutionized the field of biomedical science, enabling researchers to understand the biology behind disease mechanisms and allow the treatment of several health conditions, including ocular pathologies. The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing facilitates efficient and specific genetic modifications in the nucleic acid sequence, resulting in permanent changes at the genomic level. This approach has advantages over other treatment strategies and is promising for the treatment of various genetic and non-genetic ocular conditions. This review provides an overview of the CRISPR/CRISPR-associated protein 9 (Cas9) system and summarizes recent advances in the therapeutic application of CRISPR/Cas9 for the treatment of various ocular pathologies, as well as future challenges.

Neuroprotection of Rodent and Human Retinal Ganglion Cells In Vitro/Ex Vivo by the Hybrid Small Molecule SA-2.

The mechanisms underlying the neuroprotective effects of the hybrid antioxidant-nitric oxide donating compound SA-2 in retinal ganglion cell (RGC) degeneration models were evaluated. The in vitro trophic factor (TF) deprivation model in primary rat RGCs and ex vivo human retinal explants were used to mimic glaucomatous neurodegeneration. Cell survival was assessed after treatment with vehicle or SA-2. In separate experiments, tert-Butyl hydroperoxide (TBHP) and endothelin-3 (ET-3) were used in ex vivo rat retinal explants and primary rat RGCs, respectively, to induce oxidative damage. Mitochondrial and intracellular reactive oxygen species (ROS) were assessed following treatments. In the TF deprivation model, SA-2 treatment produced a significant decrease in apoptotic and dead cell counts in primary RGCs and a significant increase in RGC survival in ex vivo human retinal explants. In the oxidative stress-induced models, a significant decrease in the production of ROS was observed in the SA-2-treated group compared to the vehicle-treated group. Compound SA-2 was neuroprotective against various glaucomatous insults in the rat and human RGCs by reducing apoptosis and decreasing ROS levels. Amelioration of mitochondrial and cellular oxidative stress by SA-2 may be a potential therapeutic strategy for preventing neurodegeneration in glaucomatous RGCs.

Nano encapsulated novel compound SA-10 with therapeutic activity in both acute and chronic murine hindlimb ischemia models.

The production dysregulation of reactive oxygen species (ROS) and nitric oxide (NO) in ischemic tissues results in endothelial dysfunction, hyperinflammation and poor blood circulation. Here, we report a hybrid molecule, SA-10 with both NO donating and ROS scavenging abilities that demonstrated potent cytoprotection and tube formation activity in endothelial cells under H2O2-induced oxidative stress. SA-10 loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (SA-10 NPs) were delivered intramuscularly (IM) to two murine hindlimb ischemia models. In the acute mode ischemia/reperfusion (I/R), the muscle damage, hyperinflammation, and lung edema were significantly reduced 3 days post-dose while in the chronic ischemia model, significant improvement of blood perfusion and physical endurance was observed over 30 days (P < 0.05). Elderly patients with acute and chronic limb ischemia have limited options for surgical or endovascular interventions, so we anticipate that a product like SA-10 NPs has potential as one of the therapeutic alternatives to surgery.

Novel Thiol Containing Hybrid Antioxidant-Nitric Oxide Donor Small Molecules for Treatment of Glaucoma.

Oxidative stress induced death and dysregulation of trabecular meshwork (TM) cells contribute to the increased intraocular pressure (IOP) in primary open angle (POAG) glaucoma patients. POAG is one of the major causes of irreversible vision loss worldwide. Nitric oxide (NO), a small gas molecule, has demonstrated IOP lowering activity in glaucoma by increasing aqueous humor outflow and relaxing TM. Glaucomatous pathology is associated with decreased antioxidant enzyme levels in ocular tissues causing increased reactive oxygen species (ROS) production that reduce the bioavailability of NO. Here, we designed, synthesized, and conducted in vitro studies of novel second-generation sulfur containing hybrid NO donor-antioxidants SA-9 and its active metabolite SA-10 to scavenge broad-spectrum ROS as well as provide efficient protection from t-butyl hydrogen peroxide (TBHP) induced oxidative stress while maintaining NO bioavailability in TM cells. To allow a better drug delivery, a slow release nanosuspension SA-9 nanoparticles (SA-9 NPs) was prepared, characterized, and tested in dexamethasone induced ocular hypertensive (OHT) mice model for IOP lowering activity. A single topical eye drop of SA-9 NPs significantly lowered IOP (61%) at 3 h post-dose, with the effect lasting up to 72 h. This class of molecule has high potential to be useful for treatment of glaucoma.