Metabolism of digoxin and digoxigenin digitoxosides in rat liver microsomes: involvement of cytochrome P4503A

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Abstract
1. The sequential metabolism of digoxin (Dg3) to digoxigenin bis-digitoxoside (Dg2), digoxigenin mono-digitoxoside (Dg1) and digoxigenin (Dg0) was investigated in rat liver microsomes. 2. Kinetic studies produced results consistent with a single enzyme mechanism describing the successive oxidative cleavages. Formation of Dg2 was catalysed with mean (SD) Km and Vmax of 125 22 muM and 362 37/pmol/min/mg/protein, respectively. The corresponding values for the formation of Dg1 were 61 5 muM and 7 1 pmol/min/mg/protein. Dg0 formation was catalysed with the apparent values of 30 9 muM and 310 30 pmol/min/mg/protein. 3. Chemical inhibition of cytochrome P450 (CYP) 3Asubfamily with ketoconazole and triacetyoleandomycin decreased the formation of Dg2 and Dg1 by up to 90%. Antibodies specific to rat CYP3A2 lowered the rate of oxidative cleavage of Dg3 and Dg2 by up to 85%. Inhibition of CYP2E1, CYP2C subfamily and CYP1A2 by chemical and immunoinhibition did not affect initial rates of metabolism of Dg3 and Dg2. In contrast, Dg1 metabolism was not affected by triacetyloleandomycin as well as by antibodies to CYP3A2, CYP2C11, CYP2E1, CYP2B1 2B2 and CYP1A2. It was however inhibited by 80% by gestodene and 17 alpha -ethynylestradiol (selective inhibitors of human CYP3A). 4. Collectively, these data support the involvement of CYP3A in the cleavage of Dg3 and Dg2 in rat liver microsomes. The enzyme-metabolizing Dg1 remains to be identified.