Histamine H3 receptor (H3R) antagonist-Nitric Oxide (NO) donor hybrid compounds as a new therapeutic strategy for the treatment of glaucoma and retinal neuroprotection

Glaucoma is a group of degenerative disorder of the optic nerve characterized by a malfunction of ciliary processes and trabecular meshwork (TM) in the anterior chamber of the eye. This damage causes an imbalance between production and outflow of aqueous humor (AH) generating an increase of intraocular pressure (IOP). The ocular hypertension (OHT), if untreated, provokes a reduction of local perfusion and chronic ischemia leading to a depletion of retinal ganglion cells (RGCs) by autophagy and/or apoptosis. Currently, all treatment strategies are directed at lowering IOP; the two possible strategies are to reduce AH production or to increase the outflow. Unfortunately, a significant number of patients do not respond to these treatments and most of these compounds have side effects. In recent years, an approach consisting of multi-targeted compounds is emerging. This different strategy consists in design of hybrid compounds that incorporate moieties able to interact at different biological levels. Nitric oxide (NO) is an endogenous messenger that plays a role in modulating the homeostasis of IOP. Further evidence shows that a dysregulation in NO production can contribute to increasing IOP levels by promoting the development or progression of glaucoma. The histaminergic system also plays an important role in the regulation of IOP, in fact the presence of histamine H1, H3 and H4 receptors in various ocular tissues has been demonstrated. In addition, the administration of H3R antagonists has proven to be effective in reducing OHT, preventing RGCs loss by improving vascular performance of the ophthalmic artery, proposing this approach as a replacement therapy for the treatment of glaucoma. Impaired ocular blood flow has been shown to result in insufficient oxygen and nutrient supply to the RGC axons crossing the optic nerve head (ONH), causing their degeneration. Indeed, unstable ocular blood flow produced, by chronically elevated and/or oscillating IOP, together with possible vascular dysfunction can lead to repeated hypoperfusion resulting in an ischemic retinal damage. The present research was focused to evaluate the efficacy of three histamine H3R antagonist-NO donor hybrid compounds in two animal model of OHT in rabbit. Moreover, I studied the capability of one of these molecules in ameliorating vascular ocular performance and to prevent photoreceptor degeneration in a rabbit model of ischemia/reperfusion (I/R) induced by repeated injections of endothelin-1 (ET-1). The acute or chronic OHT model were obtained respectively by injection of 50 µl of hypertonic saline (5% NaCl in sterile water) into the vitreous, or 100 µl of carbomer into the anterior chamber of New Zealand White rabbits’ eye. The IOP was measured using a pneumatonometer at baseline, 60,120 and 240 minutes after OHT induction in the acute model or every day for 12 days before drug dosing in the chronic model. All animals received a topical single dose of 30 μl of studied compounds (ST-1989, ST-2126 and ST-2130) at a concentration of 1%; the molecules were compared with reference drugs ciproxifan (H3R antagonist) and molsidomine (NO donor) administered alone and in combination. The I/R model was obtained by repeated injections of ET-1 twice weekly for six weeks into the subtenon capsule of the eye. The animals were treated twice daily with vehicle or compound ST-1989 from the third week until the end of the protocol. IOP was measured every week after 36 hours of washout. Ophthalmic artery resistance index and the retinal function were assessed using an Echo Color Doppler ultrasound and Retimax Advance, respectively, at baseline, after 2 weekly injections of ET-1 and after 6 weeks. At the end of the experimental protocol, the animals were euthanized to collect AH, ciliary body, and retina tissues to perform biochemical evaluations such as the activation of NO-GC pathway (accumulation of nitrites and cGMP levels), the inflammatory response (IL-6 and TNF-α), the oxidative stress (GSH, MnSOD and 8OH2dG), and the apoptotic process (Caspase 3 activity). Furthermore, whole eyes were harvested to perform morphological and histopathological evaluations (Haematoxylin/eosin staining and TUNEL assay). Among the hybrid compounds, ST-1989 has proven to be the most responsive molecule, able to reduce the IOP in both acute and chronic OHT model demonstrating a long-lasting effect. In addition, treatment with ST-1989 was able to reduce the inflammatory response, oxidative stress, and apoptotic process in chronic OHT and attenuate hypertension, improve ocular perfusion and prevent photoreceptor degeneration in the retinal I/R model. In conclusion, these hybrid compounds could be potential therapeutic drugs for glaucoma management and retinal neuroprotection.