Increased digitoxin cleavage by liver microsomes of spironolactone-pretreated rats

Summary Pretreatment of rats with spironolactone caused an fourfold increased cleavage rate of the sugar chain of digitoxin (dt-3) in vitro yielding digitoxigenin-bis-digitoxoside. This was due to an enhanced, cyt. P450 dependent, formation of 15-dehydro-dt-3, the intermediate which has to be formed before the terminal sugar can be split off. The second reaction catalysed by microsomal monoxygenases, the 12--hydroxylation, was only increased by a factor 2.In contrast to the effects of spironolactone no increase of metabolism could be observed after phenobarbital pretreatment. From our results it may be concluded that the enhanced dt-3 metabolism in vivo is mainly caused by spironolactone inducible monoxygenases which catalyse the oxidation of the terminal sugar.     

Dehydro-digitoxosides of digitoxigenin: Formation and importance for the digitoxin metabolism in the rat

Summary Only in the presence of NADPH and oxygen digitoxosides of digitoxigenin can be metabolized by rat liver microsomes. The metabolism includes 12-hydroxylation, formation of digitoxosides less the terminal digitoxose and of lipophilic metabolites. The lipophilic metabolites of digitoxin (dt-3), digitoxigeninbis-digitoxoside (dt-2), and of — mono-digitoxoside (dt-1) are formed by oxidation of the axial OH-group of the terminal digitoxosyl yielding the corresponding dehydro-digitoxosides. The structure could be confirmed by comparison with the synthetic compounds 15-dehydro-dt-3, 9-dehydro-dt-2, and 3-dehydro-dt-1, respectively.The terminal dehydro-digitoxosyl can be split off by liver microsomes even in the absence of NADPH. However, no further cleavage of the resulting digitoxosides could be detected. A cleavage rate of 2.8 nmoles/mg microsomal protein × 30 min was observed for both 15-dehydro-dt-3 and 9-dehydro-dt-2 (substrate concentration 50 M). In contrast to that, only 0.4 nmole 3-dehydro-dt-1 was cleaved under equal conditions. For the cleavage of 15-dehydro-dt-3 an apparent K _m of 200 M and a V _max of 440 pmoles dt-2 formed per mg microsomal protein x min was measured.Our results indicate that not the native but only the dehydro-digitoxosides can be cleaved. Therefore, two successive monoxygenase catalysed oxidations are necessary for the cleavage of the sugar chain of dt-3 before dt-1, the main substrate of conjugation enzymes, can be formed. Moreover, digitoxigenin will not be formed because of the high stability of 3-dehydro-dt-1.