SAT-344 11α-Hydroxyprogesterone, a Potent 11β-Hydroxysteroid Dehydrgenase Inhibitor, Is Metabolised In Vitro by Adrenal Steroidogenic Enzymes

Abstract

The C11-oxy C21 steroids, 11αOHP4 (11α-hydroxyprogesterone) and 11βOHP4 (11β-hydroxyprogesterone), have to date been characterised only in terms of their inhibition of 11β-hydroxysteroid dehydrogenase (11βHSD). However, 11βOHP4 is also a substrate of 11βHSD2 and is converted to 11-ketoprogesterone which is subsequently metabolised to active C11-oxy androgens. Although our studies indicate that 11αOHP4 is not a substrate for 11βHSD2, it may be metabolised by the steroidogenic enzymes of the backdoor pathway. While circulating 11αOHP4 and 11βOHP4 levels are not routinely analysed in physiological and pathophysiological conditions, 11βOHP4 was only recently included in the analysis of steroid hormones associated with adrenal disorders with 11αOHP4 being overlooked. We have demonstrated that both 11αOHP4 and 11βOHP4 are present in prostate cancer tissue samples while the in vitro metabolism of 11βOHP4 contributes to the production of the potent C11-oxy C19 steroid, 11-ketodihydrotestosterone, in the C11-oxy backdoor pathway. We therefore hypothesised that 11αOHP4 would be metabolised to C11-oxy C19 steroids. The in vitro metabolism of 11αOHP4 was therefore assayed in HEK293 cells transiently transfected with 5α-reductase (SRD5A) and cytochrome P450 17A1 (CYP17A1). Ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS) analysis identified 5α-pregnan-11α-ol-3,20-dione in the conversion by SRD5A1 and SRD5A2 (50% converted in 2.5 and 1.5 hrs, respectively), the initial step in the backdoor pathway. Moreover, novel steroid metabolites were detected in the metabolism of 11αOHP4 by CYP17A1 in the production of the 11α-hydroxy derivatives of 21-deoxycortisol and 11-hydroxyandrostenedione. These were successfully confirmed by UPC2-MS/MS and chromatographically separated from the 11β-hydroxylated forms. The metabolism of 11αOHP4 and 11βOHP4 was subsequently assayed in several whole cell models in which these novel intermediates were detected as well as quantifiable levels of C11-oxy C19 steroids in the conversion of 11βOHP4. In conclusion, in vitro metabolism by SRD5A, indicates that 11αOHP4 is metabolised in the C11-oxy backdoor pathway with conversion by CYP17A1 yielding 11α-hydroxylated C19 steroids. Data suggest that 11αOHP4 may add to active C11-oxy C19 steroids with the 11α-intermediates attenuating the inhibitory potential of the steroids leading to the production of 11KDHT. However, the conversion of 11αOHP4 by SRD5A and CYP17A1 is less efficient than that of 11βOHP4—indicating that the contribution of 11αOHP4 to the active androgen pool would be markedly lower, suggesting a potential mechanism controlling the flux towards active C11-oxy androgens. Although the contribution to the androgenic pool remains unclear, the role of 11αOHP4 is more complex than that of one being labelled just as an inhibitor of 11βHSD2.