新しいステロイド合成酵素阻害剤trilostaneのin vitroにおける酵素阻害作用について

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Trilostane (WIN 24,540) blocks steroidogenesis by inhibiting the 3β-hydroxysteroid dehydrogenase system, and the effect has been produced in experimental animals and patients with certain kinds of hyperadrenocorticism. But the pharmacological profile of trilostane in the human adrenals has not been clarified, and the question exists whether or not this drug has an effect on the other steps in steroid biosynthesis. The present paper concerns the in vitro effects of trilostane on steroid production and conversion of tritiated pregnenolone to the other steroids in the adrenals. Slices of adrenocortical tissue were used for the in vitro studies. Grossly normal adrenals were obtained from 32 cattles at slaughter and surgically from a patient with breast cancer. For hyperadrenocorticism, 2 adenomas and one hyperplasia were obtained surgically from 3 patients with Cushing's syndrome, one adenoma from primary aldosteronism and one adrenal carcinoma with hypermineralocorticoidism, respectively. Tissue slices weighing about 1.0 g were placed in 50 ml of a Krebs-Ringer bicarbonate buffer (KRB) containing glucose in a concentration of 200 mg per 100 ml. Tissues were preincubated for 1 hour at 37°C under a 5% CO2-95% O2 gas mixture shaking continuously at the rate of 150 cycle/ min. After preincubation, the slices were again incubated for one hour in 50 ml of fresh KRB-glucose to which approximately 1 μCi of 7-3H-pregnenolone (Specific activity; 10 Ci/ mmol) was added with or without trilostane at the concentration from 2×10-8M to 1×10-6M. Some of the human adrenal tissues were incubated without tritiated pregnenolone for the estimation of steroid concentrations produced during the incubation. After incubation, the media were decanted, extracted with dichloromethane and dried up in a vacuum drying oven under 60°C. The steroids converted from tritiated pregnenolone were separated by a thin layer chromatography developed in a medium of absolute toluene 90 : methanol 10 and radioactivity was detected by a thin layer chromatogram scanner. Then the areas of the strip corresponding to pregnenolone, progesterone, 17α-hydroxypregnenolone and 17α-hydroxyprogesterone, corticosterone, aldosterone and cortisol were respectively cut out and eluted with methanol for the tritium content count using a liquid scintillation counter. in vitro production of steroids without tritiated pregnenolone was analyzed by radioimmunoassay after an extraction and purification by column chromatography as reported previously. The mean ratio of in vitro conversion of tritiated pregnenolone to other steroids was 65.3% (progesterone 25.7%, 17a-hydroxypregnenolone and 17α-hydroxyprogesterone 10.3%, corticosterone 22.0%, aldosterone and cortisol 4.1% and others 3.2%) of beef adrenals in the absence of trilostane. The conversion was reduced to 46.9% in the presence of 2×10-8M of trilostane and to 18.9% at the concentration of 2×10-7M of trilostane. Trilostane is an effective inhibitor of the 3β-hydroxysteroid dehydrogenase system in the beef adrenal gland. It is interesting that the doses of trilostane required to inhibit steroidogenesis in the present study were lower than those reported by Potts et al. (1978). By adding 2×10-6M of trilostane to the incubation medium for tissue slices from adrenal adenoma with Cushing's syndrome, the ratio of conversion from pregnenolone to other steroids was also reduced from 40% to 10.7% as beef adrenal slices. in vitro production of progesterone, 11-deoxycorticosterone, 17α-hydroxyprogesterone and cortisol by tissue slices from the normal adrenal cortex, aldosteronoma and adrenal carcinoma with hypermineralocorticoidism was markedly reduced by the addition of 2×10-7M of trilostane.
日本内分泌学会の論文

新しいステロイド合成酵素阻害剤trilostaneの<I>in vitro</I>における酵素阻害作用について

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