Development and application of a beta-tritium-mitotane assay for studies on the mechanism of the adrenolytic action of mitotane.

Abstract

The toxicity of mitotane limits its use in the treatment of adrenal cancer and Cushing's disease. Because of this limitation and because some patients are refractory to mitotane, our current research is directed towards the synthesis of mitotane analogs in order to produce a more effective drug. To investigate the mechanism of steroid inhibition and adrenolytic action of mitotane, an assay, based on the release of $\sp3$H$\sb2$O upon direct $\beta$-hydroxylation of $\beta$-$\sp3$H-mitotane, was developed. Kinetic studies of mitotane were performed using bovine adrenal mitochondrial acetone powder as the enzyme source. The incubation was found to be linear up to 30 min and up to 1 mg of protein. A K$\sb{m}$ of 30 $\mu$M was determined. The tritium release assay correlated with $o,p\sp\prime$-DDA formation as follow by a $\sp{14}$C-HPLC assay and is consistent with $\beta$-hydroxylation of mitotane as its mode of activation. The reaction was found to be O$\sb2$ dependent and to be inhibited by a mixture of O$\sb2$:CO (20%:80%) suggesting the involvement of a mitochondrial P-450 enzyme on mitotane transformation. A NADPH-generating system was found to be more efficient than NADPH in supporting the enzymatic reaction. The assay proved to be useful for the clinical analysis of adrenal tumors. Inhibition studies with the well described P-450 inhibitors, aminoglutethimide, metyrapone and ketoconazole, showed ketoconazole to be the most potent inhibitor of the reaction. Substrates of mitochondrial steroid synthesis, cholesterol side chain cleavage, 11$\beta$-hydroxylase and 18-hydroxylase, showed very little inhibition of enzymatic activity suggesting that none of these enzymes play a strong role on mitotane transformations. Studies of mitotane and two of its analogs as mechanism-based inactivators revealed they are poor inactivators of the enzyme responsible for mitotane metabolic activation. On the other hand, trisubstituted analogs of mitotane known to be inactive as adrenolytic agents caused a time dependent enzyme inactivation which is postulated to occur by a free radical mechanism.