In Vitro Biotransformation of 3,4-Dihydro-6-hydroxy- 2,2-dimethyl-7-methoxy-l(2H)-Benzopyran (CR-6),a Potent Lipid Peroxidation Inhibitor and Nitric Oxide Scavenger,in Rat Liver Microsomes

摘要

The in vitro metabolism of 3,4-dihydro-6-hydroxy-2,2-dimethyl-7-methoxy-l(2H)-benzopyran (CR-6),a potent lipid peroxidation inhibitor and scavenger of nitric oxide and peroxynitrite species that is currently in phase II trials for antitumoral therapy,has been investigated in rat liver microsomes in the presence of NADP(H).Five major metabolites were identified by comparison with authentic standards,namely,the quinone 2-(3'-hydroxy-3'-methylbutyl-5-methoxy-l,4-benzoquinone (2a) and its ring-closed spiro form oxaspiro[4.5]-2,2-dimethyl-8-methoxy-dec-8-ene-7,10-dione (2b),the hydroquinone 2-(3'-hydroxy-3'-methylbutyl)-5-meth-oxyhydroquinone (3),the hydroxylated metabolite 3,4-dihydro-4,6-dihydroxy-2,2-dimethyl-7-methoxy-l(2H)-benzopyran (4),and the catechol 3,4-dihydro-6,7-dihydroxy-2,2-dimethyl-l(2H)-benzopyran (5).When the incubations were carried out in the presence of GSH,the HPLC peaks corresponding to the quinone metabolites 2a/b were absent and two novel products were formed showing MS fragmentation patterns consistent with the structure of GSH conjugates of quinone 2a.The time dependence on the formation of metabolites 2a,b and 3 was measured in incubations induced with phenobarbital (PB),dexamethasone,and beta-naphthoflavone (betaNF).For the dexamethasone-induced microsomes,the amount of hydroquinone 3 decreased from minute 10 to minute 30 while that of 2a,b increased in a complementary manner.Similar effects were observed for the incubations carried out using PB- and betaNF-induced microsomes.On the other hand,CR-6 inhibited 7-ethoxyresorufin O-dealkylation activity (IC_(50)=25 muM) in incubations with /?NF-induced microsomes.Likewise,addition of pentoxyresorufin to the incubations of CR-6 with PB-induced microsomes showed a time-dependent inhibition (IC_(50)=75 muM) of the dealkylation activity.These results are in agreement with the putative generation of reactive metabolites from CR-6 that could deactivate P450 1A and P450 2B,respectively.When these incubations were carried out in the presence of 10 mM GSH,the inhibition of P450 2B could be partially prevented.Finally,preincubation of CR-6 with liver microsomes from PB-induced rats resulted in a strong increase in microsomal glutathione S-transferase (mGST) activity (up to a maximum of approximately 5-fold).When the preincubation was carried out in the presence of 10 mM GSH,the activation of mGST was blocked.Overall,these results suggest that CR-6 undergoes in vitro biotransformation indicative of the involvement of thiol-reactive metabolites.