Abstract
Purpose: :
Senile cataracts are strongly associated with postsynthetic modifications of lens crystallins by advanced ascorbylation end products that are known to enhance crystallin aggregation and light scattering. Guanidino compound NC1 fed to hSVCT2 mice was previously found to block ascorbylation reactions (Fan et al, IOVS 49:4945-52,2008). As a preamble to the topical application of NC1 as a drug applied to the human eye, we have compared the uptake of NC1 and N-acetyl-cysteine (NAC) in rabbit lens in vitro and in vivo, as well as the ability of NC1 to block ascorbylation when applied topically to hSVCT2 mouse model of lenticular aging.
Methods: :
Rabbit lenses (n=8) were incubated for 24 hr with each 5 mM NC1 and NAC in presence of ascorbic acid (5 mM), dehydroascorbic acid (5 mM), or glucose (25mM) for 24 hrs at 37[[Unsupported Character - ◦]]C. NC1 and NAC uptake in the protein-free fraction was determined in the water soluble fraction with a Micromass Ultima LC/MS instrument. Rabbits (n=8) also received 0.5% and 1.0% NC1 and NAC applied to each eye and uptake was determined during a 4 hour period.hSVCT2 mice (n=10/group) were treated with 2 drops/day of 1% NC1 and 1% NAC in each eye for 7 months. Advanced glycation endproducts were measured in the insoluble protein fraction as reported earlier (Fan et al, JBC Oct 23, 2009).
Results: :
In rabbit lenses incubated with NC1, levels increased 4-5 fold to 31.5 ±8.3 µmol/g in all incubations, while NAC increased to only 0.03±0.0018 µmol/g, indicating high affinity uptake of NC1. In rabbit’s eye treated topically with 0.5 and 2.0% NC1 reached identical plateau (18 +/- 2.5µmole/g) after two hours and returned to baseline after 5 hours at 0.5%, but remained at 80% at 2% concentration. NC1 applied topically to hSVCT2 mice decreased protein-bound 335/385 nm fluorescence and the glycation products CML, CEL and glucosepane crosslinks by 40% (p<0.001), 35% (p<0.05), 30% (p<0.05) and 37% (p<0.05, ), respectively, without affecting MG-H1 and G-H1 hydroimidazolones levels. NAC decreased fluorescence by 50%, while penicillamine and guanidino compound NC2 (IOVS 49:4945-52, 2008) had no effect.
Conclusions: :
Guanidino compound NC1 is readily taken up via topical application to the lens, in contrast to NAC which suppressed only fluorescence. This, with the broad ability of NC1 to inhibit undesirable crystallin modifications, opens up the possibility of testing the anticataract potential of NC1 in clinical trial.
Keywords: cataract • aging • oxidation/oxidative or free radical damage