In vitro activity: Aminoglutethimide displays aromatase inhibition in vitro assay with human placental aromatase, which is an enzyme involved in the conversion of androgens into estrogens, and an important target for the endocrine treatment of breast cancer. Aminoglutethimide inhibits ACTH receptor (ACTH-R) mRNA expression in ovine adrenocortical cells in a time-dependent fashion. Aminoglutethimide significantly suppresses steroid secretion and the baseline ACTH-R mRNA expression in a dose-dependent fashion (300 μM AG, 5±1%; 30 μM AG, 64±1%; 3 μM AG, 108±19% compared with control cells, 100±11%) by affecting the gene expression or by decreasing transcript accumulation via an effect on RNA stability, in the human NCI-h295 adrenocortical carcinoma cell line, which expresses functional ACTH receptors and produces steroids of the glucocorticoid, mineralocorticoid and androgen pathway. Aminoglutethimide inhibits aromatase in a dose-dependent fashion with IC50 of 13 μM in 6 breast tumor homogenates, placental aromatase with IC50 of 6 μM and hypothalamic aromatase with IC50 of 8 μM.

Kinase Assay: The microsomal protein (30 μg), [1β-3H]androstenedione (6.6 × 105 dpm) and NADPH (270 μM) are used for the concentration–response experiment with an incubation time of 20 minutes. The Aminoglutethimide is initially tested at 10 μM and 100 μM concentrations, followed by a full concentration–response study with at least 8 concentrations ranging from 0.01 μM to 160 μM. For the initial velocity study the concentration of [1β-3H]androstenedione is varied from 7.5 to 100 nM and the incubation time is set to 5 minutes. The tritiated water formed during the conversion of the tritiated substrate, [1β-3H]androstenedione, to estrone is quantified by liquid scintillation counting. Each assay is performed three times in duplicate and the results are treated by nonlinear regression analysis allowing the determination of the half-maximal inhibitory concentration (IC50).

Cell Assay: The NCI-h295 tumor cell line is maintained in RPMI 1640 medium supplemented with transferrin (0.1 mg/mL), insulin (5 μg/mL), selenium (5.2 μg/mL) and 2% FCS. The cells are incubated for 48 hours with Aminoglutethimide (3, 30, 300 μM). Then cells are examined by trypan blue staining for cell viability, counted with a coulter counter. For the assessment of ACTH-R mRNA, cells are harvested, and total RNA is extracted, electrophoresed, blotted and hybridized with a human ACTH-R cDNA probe.

Eur J Med Chem. 2009 Oct;44(10):4121-7; Cancer Res. 1982 Aug;42(8 Suppl):3353s-3359s.