Fig 1.
PBND tested for RPE-65 enzyme inhibition efficiency.
The structure of PBN and the derivatives of PBN that are used in various in vitro and in vivo assays are shown in Fig 1A–1D. Concentration-dependent inhibition of RPE65 and generation of 11-cis-retinol in in vitro microsome assays were performed with representative images shown in Fig 1E, 4-F-PBN; Fig 1F, 4-CF3-PBN; and Fig 1G, 4-CH3-PBN. A table of the IC50 values of the compounds tested for their inhibition efficiency for RPE 65, (Fig 1J), show various inhibition rates for which we determined which PBN-derivatives looked the most promising to continue our analysis.
Fig 2.
PBN and certain PBNDs inhibited rhodopsin regeneration.
Rats were treated with either PBN or certain PBNDs before 2h of light adaptation followed by 2.5h of dark adaptation after which the animals were euthanized and retinas harvested for rhodopsin assay. Saline treated retina recovered ~ 75% rhodopsin in 2.5h dark. PBN and its derivatives, however, blocked rhodopsin regeneration significantly. LA, light adapted; DA, dark adapted. [* = p<0.01; ** = p<0.001; n = 4–6]
Fig 3.
Systemically administered PBN and PBND protect rat retina from light damage.
Electroretinographic (ERG) responses were recorded for a series of flash stimuli intensities at 0.04, 4, 200, 400, and 2000 cd.sec/m2. ERG responses presented here are from the intensities of 4 and 400 cd.sec/m2. The dim flash (4 cd.sec/m2) stimulates the rod photoreceptor cells and the bright flash (400 cd.sec/m2) stimulates both rod and cone photoreceptors. Therefore, the blue bars represent only rod responses and the red bars represent mixed response from both rod and cone photoreceptors. Fig 3A shows A-wave responses, and Fig 3B shows B-wave responses. NLD represents non-light-damaged group and LD represents light-damaged groups. [## = p<0.001: NLD vs. LD saline-treated; * = p<0.01 and ** = p<0.001: LD saline vs. PBNDs; n = 4–6]
Fig 4.
Histological analysis of rat retina confirms protection from light damage.
Vehicle or Saline treated (Vehicle LD) rats lost most of their photoreceptors from the central retina. Effect is more pronounced in the superior retina. NLD is non-light-damage control. Fig 4A shows the ONL thickness across the retina in the vertical meridian. PBN treatment (PBN LD) retinas retained ~90% of photoreceptor cells. 4-CF3-PBN is comparable to PBN in protection, whereas 4-F-PBN appears better than PBN. 4-CH3-PBN also protects the retina significantly, although its effect is slightly less than PBN. Fig 4B shows central retinal outer retinal layer (ONL) thickness in both inferior and superior retina. [** = p<0.001; n = 8–10]
Fig 5.
Mice treated topically with PBN or PBND’s showed reduction in rhodopsin regeneration.
Dark adapted no treatment (DA-NT), no treatment light-adapted followed by dark adapted (NT LA-DA), and vehicle controls were used. Both 1% and 10% PBN were tested against 5% concentrations of PBNDs. Topical administration of assigned drug consisted of 1 drop in each eye once every 3 hours over a 6 hour period resulting in 3 total applications in light adapted mice eyes. Immediately following the 3rd and final dose mice were placed in the dark for 1h before harvest. LA, light adapted; DA, dark adapted. [* = p<0.01; ** = p<0.001; n = 6]
Fig 6.
PBN and 4-CH3-PBN topically administered in baboon eyes slows rate of rhodopsin regeneration.
Baboon eyes were treated topically with drug or vehicle every 15 minutes over a 2h period before eyes were harvested and dark adapted 2h before retinal harvest. 10% PBN showed a roughly 50% higher inhibition rate compared to 5% CH3-PBN suggesting both drugs to be effective. [** = p<0.001; n = 6–8 eyes]