Evaluation of DNA damage in spinal cord and mutagenic effect of a Phα1β recombinant toxin with analgesic properties from the Phoneutria nigriventer spider

Abstract

Phα1β peptide isolated from the venom of the Phoneutria nigriventer spider has shown higher analgesic action in pre-clinical studies than ω-conotoxin MVIIA peptide used to treat severe chronic pain. In view of the great potential for the development of a new Phα1β-based drug, a Phα1β recombinant form (CTK 01512-2) has been studied for efficacy and safety. The aim of this study was to evaluate cytotoxic, genotoxic and mutagenic effects of a Phα1β recombinant form and compare it with native Phα1β and ω-conotoxin MVIIA. Cytotoxicity was evaluated using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colourimetric assay in L929 mouse fibroblast cells (0.5-10.0μmol/L). Genotoxic and mutagenic activities were analysed using the alkaline comet assay in peripheral blood and spinal cord, and the micronucleus test in bone marrow from Wistar rats treated by intrathecal injection of CTK 01512-2 (200, 500 and 1000pmol/site), native Phα1β (500pmol/site) and ω-conotoxin MVIIA (200pmol/site). CTK 01512-2 decreased the cell viability of the L929, showing IC50 of 3.3±0.1µmol/L, while the Phα1β and ω-conotoxin MVIIA did not show cytotoxicity (IC50 >5.0µmol/L). Native and recombinant Phα1β forms induced DNA damage in the spinal cord, but not in peripheral blood. CTK 01512-2 at 1000pmol/site increased the micronucleus frequency suggesting mutagenic effects. In conclusion, the recombinant form has cytotoxic, genotoxic and mutagenic effects, evidenced in doses five times above the therapeutic dose.