Drug Design of Mitochondria-Targeted Antioxidants, Action, Metabolism and Perspectives for Ophthalmic Therapeutics: N-acetylcarnosine Codrug Treatment Platform

Maintaining the redox balance within the mitochondria is critical for cellular homeostasis in the eye since the mitochondria host the energy producing systems of the cell and it is widely recognized that damage to the mitochondria plays a key role in sight threatening age-related eye disorders, including retinopathies (maculodystrophy, retinitis pigmentosa , hereditary optic neuropathy), as well as glaucoma, cataract, and autoimmune uveitis.

Reactive oxygen species (ROS) are generated as by-products of cellular metabolism, primarily in the mitochondria. Although ROS are essential participants in cell signaling and regulation, when their cellular production overwhelms the intrinsic antioxidant capacity, damage to cellular macromolecules such as DNA, proteins, and lipids ensues.

Oxidized phospholipids play an important role in execution of the mitochondrial stage of apoptosis and clearance of apoptotic cells. During the lipid peroxidation (LPO) reaction, lipid hydroperoxides are formed as primary products. Several lines of evidence suggest that lipid hydroperoxides can trigger cell death in many cell types, which may be mediated by mitochondria dysfunction pathway. Recently, there was a breakthrough in mitochondrial targeting of antioxidants. Mitochondrial function can be manipulated selectively by targeting bioactive compounds to mitochondria in living cells. Lipophylic antioxidants were covalently coupled to a triphenylphosphonium cation, and these compounds were preferentially taken up by mitochondria.

new pharmacologic tool to mitigate complex ocular pathology for the treatment of sight-threatening eye diseases

 

The effect of carnosine on self-organization of mitochondrial assemblies was studied in rat liver homogenate of quiescent and excited animals. It was shown in separate electron microscopy experiments with serial slices that under our conditions of preparation of homogenate, blocks of native mitochondrial-reticular net work in the cell, assemblies of mitochondria, are kept. Carnosine was shown to prevent dissociation of assemblies during storage. Its effect is maximal for more dissociated assemblies from excited animals with decreased ability for self-organization.

Recently, phospholipid peroxidation products gained a reputation as key regulatory molecules and participants in oxidative signaling pathways. Oxidation of two anionic phospholipids–cardiolipin (CL) in mitochondria and phosphatidylserine (PS) in extramitochondrial compartments–is important signaling event, particularly during the execution of programmed cell death and clearance of apoptotic cells. Quantitative analysis of CL and PS oxidation products is central to understanding their molecular mechanisms of action.

In this work we proposed the combined use of mitochondria-targeted antioxidant mito Vit E and N-acetylcarnosine, an ophthalmic prodrug of L-carnosine in formulation of eye drops including the mucoadhesive compound carboxymethylcellulose to help elucidate the role of mitochondrial oxidative damage in apoptotic cell death.

We suggest that mitochondrial oxidative damage plays an important role in ROS-induced apoptosis. Further work using these and other mitochondrially targeted compounds to dissect out the role of mitochondrial oxidative changes in peroxide-induced apoptosis is ongoing. The findings reported here demonstrate that mitochondrially targeted antioxidants such as mito vit E + N-acetylcarnosine in the eye drop formulation with carboxymethylcellulose can be used to investigate the role of mitochondrial oxidative stress in RGCs cell death. This strategy also has potential for unraveling the contribution of oxidative stress to other ocular pathologies involving mitochondrial dysfunction.

With the recognition of the central role of mitochondria in apoptosis, there is a need to develop specific tools to manipulate mitochondrial function within cells in ocular disorders, such as glaucoma, human cataract and retinal disorders. Here we report on the development of a novel antioxidant eye drop formulation N-acetylcarnosine eye drop prodrug–codrug as an ocular promoter of L-carnosine, a natural mitochondria-targeted antioxidant, activated by a triphenylphosphonium cation coupled bioactive molelcules delivered to mitochondria that selectively blocks mitochondrial oxidative damage, enabling the roles of mitochondrial oxidative stress in different types of ocular cell death to be inferred .

Due to the large mitochondrial membrane potential, the cation was accumulated within mitochondria inside cells, where the lipophilic moiety of mitochondria-targeted antioxidant inserted into the lipid bilayer and was reduced by the respiratory chain. Due to the combination of weak metal chelating (abolished by EDTA), OH˙ and lipid peroxyl radicals scavenging, reducing activities to liberated fatty acid and phospholipid hydroperoxides, carnosine derived in the ocular tissues and fluids from the ophthalmic prodrug N-acetylcarnosine, appears to be a physiological universal antioxidant able to efficiently protect the lipid phase of biological membranes and aqueous environments and act as the anti-apoptotic natural drug compound . After detoxifying a reactive oxygen species, the lipophilic moiety of mitochondria-targeted antioxidant was regenerated by the respiratory chain enabling its antioxidant activity to be recycled. We have shown that selectively manipulating mitochondrial antioxidant status with targeted dipeptide and recyclable antioxidants is a feasible approach to investigate the role of mitochondrial oxidative damage in apoptotic cell death in sight-threatening eye disorders. This approach will have further clinical applications in investigating mitochondrial dysfunction in a range of experimental models.

We conclude that, based on the data presented in this study, the combined eye drop formulation including N-acetylcarnosine prodrug–codrug as an ocular promoter of L-carnosine, a natural mitochondria-targeted antioxidant , activated by a triphenylphosphonium cation coupled bioactive molelcules delivered to mitochondria that selectively block mitochondrial oxidative damage, may represent new pharmacologic tool to mitigate complex ocular pathology for the treatment of sight-threatening eye diseases and, especially, neurodegeneration originating from an oxidative injury and glaucomatous neurodegeneration.

published by SciDocPublishers

 

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