SUN-242 - Adrenocortical cell model allowing rapid testing of aldosterone synthase inhibitors.

Background: The use of aldosterone synthase inhibitors (ASIs) as alternative treatment options to mineralocorticoid receptor antagonists for excess aldosterone diseases is a growing focus of research and industry. ASIs directly target aldosterone synthase (CYP11B2), which converts 11-deoxycorticosterone (DOC) to aldosterone. In vitro testing is a key early-stage step in evaluating ASI efficacies. The human adrenocortical carcinoma cell line, NCI-H295R (H295R) is widely used to study adrenal cell steroid hormone production and recently, for ASI testing; however, steroidogenesis of this model varies from laboratory to laboratory with some reporting low aldosterone production. As a result, ASI testing requires chronic experiments and stimulation with agonists to ensure sufficient accumulation of aldosterone. Here, we test three clonal cell lines derived from H295R and their potential to act as an alternative in vitro CYP11B2-expressing cell model and the ability of the aldosterone precursor (DOC) to enhance aldosterone production and allows acute ASI testing.

Methods: The H295R-derived clonal cell lines (HAC13, HAC15, and HAC50) were cultured and seeded into 48-well plates (50,000 cells/well). After 48h, cells were incubated with and without the aldosterone precursor, DOC (10 µM), for 1, 2, 3, 4, and 6h followed by measurement of medium aldosterone. ASI activity was examined in the HAC13, HAC15 and HAC50 clones using three ASI: Osilodrostat (30 nM), Baxdrostat (300 nM), and Lorundrostat (300 nM) in 3h incubations with and without DOC. Medium aldosterone was quantified using an ELISA.

Results: While basal levels of aldosterone production were detectable in all cell models, DOC incubation caused a time-dependent increase in aldosterone that was significant within 1h (6.1-fold; P< 0.0001). Aldosterone production increased in HAC13, HAC15, and HAC50 cells following a 3h incubation with DOC compared to cells incubated without DOC (5.3-, 8.9-, and 8.0-fold, respectively; P< 0.001, P< 0.0001, and P< 0.0001, respectively). In contrast, DOC-enhanced aldosterone production in HAC13, HAC15, and HAC50 cells treated with ASI demonstrated a significant decrease in aldosterone output compared to cells treated with DOC alone (Osilodrostat and Baxdrostat: 0.2-fold for each cell line; Lorundrostat: 0.3-, 0.3-, and 0.4-fold, respectively; P< 0.0001).

Conclusion: Incubation of adrenal HAC clonal cell lines with DOC acutely enhanced aldosterone production. This approach shortened experimental times needed for testing ASI effects on aldosterone production. These findings support the potential for adrenal cell DOC precursor incubation as a practical and easily applicable approach for early-stage in vitro ASI screening.

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