PBN (Phenyl-N-Tert-Butylnitrone)-Derivatives Are Effective in Slowing the Visual Cycle and Rhodopsin Regeneration and in Protecting the Retina from Light-Induced Damage

Abstract

A2E and related toxic molecules are part of lipofuscin found in the retinal pigment epithelial (RPE) cells in eyes affected by Stargardt’s disease, age-related macular degeneration (AMD), and other retinal degenerations. A novel therapeutic approach for treating such degenerations involves slowing down the visual cycle, which could reduce the amount of A2E in the RPE. This can be accomplished by inhibiting RPE65, which produces 11-cis-retinol from all-trans-retinyl esters. We recently showed that phenyl-N-tert-butylnitrone (PBN) inhibits RPE65 enzyme activity in RPE cells. In this study we show that like PBN, certain PBN-derivatives (PBNDs) such as 4-F-PBN, 4-CF3-PBN, 3,4-di-F-PBN, and 4-CH3-PBN can inhibit RPE65 and synthesis of 11-cis-retinol in in vitro assays using bovine RPE microsomes. We further demonstrate that systemic (intraperitoneal, IP) administration of these PBNDs protect the rat retina from light damage. Electroretinography (ERG) and histological analysis showed that rats treated with PBNDs retained ~90% of their photoreceptor cells compared to a complete loss of function and 90% loss of photoreceptors in the central retina in rats treated with vehicle/control injections. Topically applied PBN and PBNDs also significantly slowed the rate of the visual cycle in mouse and baboon eyes. One hour dark adaptation resulted in 75–80% recovery of bleachable rhodopsin in control/vehicle treated mice. Eye drops of 5% 4-CH3-PBN were most effective, inhibiting the regeneration of bleachable rhodopsin significantly (60% compared to vehicle control). In addition, a 10% concentration of PBN and 5% concentration of 4-CH3-PBN in baboon eyes inhibited the visual cycle by 60% and by 30%, respectively. We have identified a group of PBN related nitrones that can reach the target tissue (RPE) by systemic and topical application and slow the rate of rhodopsin regeneration and therefore the visual cycle in mouse and baboon eyes. PBNDs can also protect the rat retina from light damage. There is potential in developing these compounds as preventative therapeutics for the treatment of human retinal degenerations in which the accumulation of lipofuscin may be pathogenic.

Para-Substituted α-Phenyl-N-tert-butyl Nitrones: Spin-Trapping, Redox and Neuroprotective Properties

“…This indicates the peculiar properties of this derivative. Previous work showed that 4-CF 3 -PBN efficiently protected rat retina from light damage when administered intraperitoneal . Our data therefore confirm the potency of 4-CF 3 -PBN to reduce oxidative stress and cell death.…”

“…Moreover, linear PBN-type nitrones allow the possibility of rather easy functionalization both on the aromatic ring and on the N - tert -butyl group and therefore provide more chemical versatility. Several studies showed that PBN-type nitrones combine anti-inflammatory, antioxidant, and neuroprotective properties and are able to cross the blood–brain barrier. , These nitrones could therefore be used for the treatment of stroke, visual loss, , neuronal damage, and other age-related diseases . The PBN derivative called 2,4-disulfophenyl- N - tert -butyl nitrone (NXY-059) was the first neuroprotective agent to reach phase III clinical trials in the United States. , …”

“…Nitrones have a very broad activity that depends on the nature and the position of the substituents on the nitronyl function. Therefore, the choice of substituents is very important and depends on the properties to be improved such as water solubility, , lipophilicity, , rate constant of radical trapping, − adduct stability, bioactivity, , and the possibility of cellular or tissue targeting thanks to the ligation to specific molecular targets. − Over the past several years, the reactivity of the para-substituted derivatives of PBN has been explored to identify the most promising substituent for improved and optimal reactivity toward free radicals. It has been shown that the electronic nature of the substituent influences the rate of radical trapping on the nitronyl function.…”

Low-Luminance Blue Light-Enhanced Phototoxicity in A2E-Laden RPE Cell Cultures and Rats

“…We found that BLL causes fundus damage, total retinal thickness decrement, photoreceptor atrophy, and neuron transduction dysfunction in a BN rat model [30]. Meanwhile, A2E is a major component in lipofuscin, which is a well-known metabolic deposit between the choroidal and RPE layers [2]. There are currently no effective treatments for dry-AMD, and many unknown mechanisms regarding BLL/A2E-induced photo-toxicity need to be elucidated before pre-clinical animal models can be established.…”

“…Abnormal regeneration of the visual retinoid (retinaldehyde) chromophores—between photoreceptor cells and retinal pigment epithelial (RPE) cells—non-enzymatically forms lipofuscin, thereby threatening the survival of retinal cells. These chromophores are involved in the pathogenesis of retinal degenerative diseases such as age-related macular degeneration (AMD) and Stargardt’s disease (STGD) [1,2]. Dry (atrophic) AMD accounts for most AMD cases, and is characterized by RPE degeneration and damage followed by atrophy and diminished nearby photoreceptors, causing retinal thinning and even vision loss [3].…”