Detection of Diapocynin and Apocynin Accumulation in Low Micromolar Concentrations in the CNS by HPLC-MS using Selective Ion Monitoring

摘要

NADPH oxidase has recently been identified as a promising new therapeutic target in ALS. Genetic deletion of NADPH oxidase (Nox2) in the transgenic SOD1~(G93A) mutant mouse model of ALS was reported to increase survival remarkably by 97 days. Furthermore, apocynin, a widely used inhibitor of NADPH oxidase, was observed to dramatically extend the survival of the SOD1~(G93A) ALS mice even longer to 113 days (Harraz et al. J Clin Invest 118: 474, 2008). Diapocynin, the covalent dimer of apocynin, has been reported to be a more potent inhibitor of NADPH oxidase. We compared the protection of diapocynin to apocynin in primary cultures of SOD1~(G93A)-expressing motor neurons against nitric oxide-mediated death. Diapocynin (10 (mu)M) provided significantly greater protection than apocynin 200 (mu)M at the lowest statistically significant concentrations. However, administration of diapocynin starting at 21 days of age in the SOD1~(G93A)-ALS mouse model did not extend lifespan. Repeated parallel experiments with apocynin failed to yield protection greater than a 5-day life extension in multiple trials conducted at two separate institutions. The maximum protection observed from any treatment was an 8-day extension in survival when diapocynin was administered at 100 days-of-age at disease onset. HPLC with selective ion monitoring by mass spectrometry revealed that both apocynin and diapocynin accumulated in the brain and spinal cord tissue to low micromolar concentrations. Diapocynin was also detected in the CNS of apocynin-treated mice. Although apocynin was well-tolerated natural product, the failure to achieve protection with either of apocynin or diapocynin raise questions about the utility for treating ALS patients.