Development of CYP11B1 and CYP11B2 assays utilizing homogenates of adrenal glands: Utility of monkey as a surrogate for human

• Published in: The Journal of steroid biochemistry and molecular biology 2015-11, Vol.154, p.197-205
• Main Authors: Cerny, Matthew A., Csengery, Alexander, Schmenk, Jennifer, Frederick, Kosea
• Format: Article
• Language: English
• Subjects: Adrenal Glands - enzymology; Aldosterone; Aldosterone synthase; Animals; Cortisol; CYP11B1; CYP11B2; Cytochrome P-450 CYP11B2 - antagonists & inhibitors; Cytochrome P-450 CYP11B2 - metabolism; Cytochrome P450; Humans; Inhibitors; Kinetics; Macaca fascicularis; Models, Animal; Steroid 11-beta-Hydroxylase - antagonists & inhibitors; Steroid 11-beta-Hydroxylase - metabolism
• ISSN: 0960-0760; 1879-1220
• DOI: 10.1016/j.jsbmb.2015.08.004

[Display omitted] •CYP11B kinetics and inhibition was assessed using homogenates of adrenal glands.•Similarity between monkey and human CYP11B was demonstrated.•Comparable kinetics was observed for CYP11B1- and CYP11B2-mediated reactions.•Inhibition of human and monkey CYP11B1 and CYP11B2 showed good correlations inhibitors.•Cynomolgus monkey CYP11B assays are suitable surrogates for the human enzymes. Elevated levels of aldosterone are associated with arterial hypertension, congestive heart failure, chronic kidney disease, and obesity. Aldosterone is produced predominantly in the zona glomerulosa of the cortex of the adrenal gland by the enzyme aldosterone synthase (CYP11B2). Treatment of the above indications by decreasing production of aldosterone is thought to be of therapeutic benefit by lessening the deleterious effects of aldosterone mediated through both the mineralocorticoid receptor and also through so called non-genomic pathways. However, inhibition of the highly similar enzyme, CYP11B1, which is responsible for the production of cortisol, must be avoided in the development of clinically useful aldosterone synthase inhibitors due to the resulting impairment of the cortisol-induced stress response. In efforts to assess the interactions of compounds with the CYP11B enzymes, a variety of cell-based inhibitor screening assays for both CYP11B1 and CYP11B2 have been reported. Herein we report details of assays employing both cynomolgus monkey adrenal homogenate (CAH) and human adrenal homogenate (HAH) as sources of CYP11B1 and CYP11B2 enzymes. Utilizing both CAH and HAH, we have characterized the kinetics of the CYP11B1-mediated conversion of 11-deoxycortisol to cortisol and the CYP11B2-mediated oxidation of corticosterone to aldosterone. Inhibition assays for both CYP11B1 and CYP11B2 were subsequently developed. Based on a comparison of human and monkey amino acid sequences, kinetics data, and inhibition values derived from the HAH and CAH assays, evidence is provided in support of using cynomolgus monkey tissue-derived cell homogenates as suitable surrogates for the human enzymes.