Subject: Inhibitors of CYP11B enzymes, particularly of aldosterone synthase, are currently under development as adrenostatic agents in heart failure or hyperaldosteronism, and for use in adrenal imaging. As CYP11B1 and CYP11B2 show high homology, analysis of specific binding to the respective human enzymes is of key importance.
Methods: Y1 cell lines stably expressing hsCYP11B1 or hsCYP11B2 were established. Primary cultures of porcine and human adrenocortical tissue were prepared. To assess the comparability of different systems NCI-h295 cells, transfected Y1 cells, and primary cultures were treated with different CYP11B inhibitors. Measurement of aldosterone and cortisol in the supernatant was performed.
Results: Potency as well as specificity of the tested inhibitors to CYP11B enzymes showed substantial differences in the respective test systems. The determined IC50 values of several inhibitors tested in porcine primary cultures were an order of magnitude higher compared to human primary cultures. In contrast, NCI-h295 and hsCYP11B1 and CYP11B2 expressing Y1 cells led to results similar to those from primary cultures of human adrenal glands. For example, IC50 values for etomidate for inhibition of Cyp11B1: porcine primary cultures 69.4±0.7 nmol/l; human primary cultures 5.6±1.5 nmol/l; NCI-h295 cells 4.7±1.5 nmol/l; Y1-HsCyp11B1 cell line 1.1±0.7 nmol/l.
Conclusion: Due to the poor availability of human adrenal glands for primary cultures as well as their limitations due to their continuous loss of activity, a comparable test-system for CYP11B inhibitors is strongly required. Due to the presence of both CYP11B enzymes within the same cell and potential interaction of steroidal intermediates, NCI-h295 cells are of limited value for selectivity testing. Y1 cell lines expressing human CYP11B1 and CYP11B2 enzymes, respectively, are suitable and easy to use systems for screening of inhibitors.
03 - 07 May 2008
European Society of Endocrinology