ournal.pone.0123122.t001 compound 30 from 7 to 56 dpi. Interestingly, we showed a time-dependent effect of EGCG and compound 30 in the prevention of neuropathic pain measured by thermal stimuli. We observed an increase in paw withdrawal latency in CCI-mice Danoprevir chemical information treated with EGCG from 7 to 21 dpi, with a greater effect at 7 dpi. In the other side, CCI-mice treated with the Fig 1. Administration of EGCG and compound 30 produce a time-dependent increase in CCI-mediated thermal paw withdrawal latency. The latency to paw withdrawal to a thermal stimulus was recorded in CCImice treated with vehicle and 50mg/Kg of EGCG, compound 23 and compound 30 from 7 to 56 days post injury. The results are expressed in seconds and data shown are the mean SEM. Data were analyzed by two-way ANOVA with Bonferroni’s post-hoc test. p<0.001 compared to vehicle-treated CCI-mice. doi:10.1371/journal.pone.0123122.g001 6 / 15 A Novel EGCG Derivative for Reducing Neuropathic Pain after CCI Fig 2. EGCG and compound 30 inhibit FASN activity in the spinal cord of CCI-mice. The FASN activity was analyzed in the dorsal horn of the spinal cord of CCI-mice treated with vehicle, and 50 mg/Kg of EGCG, compound 23 and compound 30 as indicated in materials and methods at 14 and 56 days post injury. Data were expressed as a percentage with respect to vehicle-treated CCI-mice and represent the mean SEM. Data were analyzed by one-way ANOVA with Bonferroni's post-hoc test. p<0.05 and p<0.001 compared to vehicle-treated CCI-mice. Protein extracts from the dorsal horn of the spinal cord of control mice and CCI-mice treated with vehicle, EGCG, compound 23 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19769484 and compound 30 at 14 and 56 dpi were subjected to western blot to study the FASN protein levels. Representative immuno-blots showing no differences in FASN levels between all groups are presented. Actin levels were PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19769788 used as loading control. doi:10.1371/journal.pone.0123122.g002 compound 30 resulted in an augmentation in the paw withdrawal latency from 14 to 56 dpi, with a highest effect from 35 to 56 dpi. Concerning the compound 23, we did not detect differences at any time point analyzed. Stability studies of EGCG and compounds 23 and 30 showed that half-life of compound 23 in human and mouse serum is 1,40,3 and 0,3 0,1 hours, respectively and regarding compound 30, 1,90,2 and 0,40,3 hours in human and mouse serum. EGCG half-life 
in human and mouse serum is inferior to 0,1 hour. In serum, compound 23 and compound 30 are in equilibrium with their metabolites, which also displayed strong FASN activity inhibition, in particular compound 30. In addition, metabolites showed similar stability as their parental compounds 23 and 30. The prevention of CCI induced-neuropathic pain by EGCG and compound 30 is associated to a reduction of FASN activity in the dorsal horn of the spinal cord It has been well reported the role of fatty acids in neuropathic pain after peripheral nerve injuries. As it has been described in non-neural cells that EGCG inhibits the fatty acid synthase activity , we studied the inhibition of FASN activity in the dorsal horn of the spinal cord of CCI-mice treated with EGCG and the two polyphenolic synthetic derivatives at 14 and 56 dpi. At 14 dpi, we observed that the administration of 50 mg/Kg of EGCG and the compound 30 reduced significantly the FASN activity to 578 and 618% of vehicle group. No differences were detected in CCI-mice administrated with the compound 23. Importantly, the reduction of FASN activity at 56 dpi was