Pharmaceutical-induced disturbances of adrenal steroidogenesis with a focus on pseudohyperaldosteronism: Identification and characterization of potentially hazardous drugs

Adrenal steroidogenesis has long been neglected in preclinical toxicity evaluations even though it is a common toxicological target. Unwanted interferences with steroidogenic enzymes increase the risk for unexpected side effects, thus posing a risk for patients. Examples of previously insufficiently tested drugs interfering with the activity of steroidogenic enzymes of the adrenal cortex are posaconazole and itraconazole, both leading to secondary hypertension and hypokalemia via pseudohyperaldosteronism.
Inhibition of 11β-hydroxylase (CYP11B1) and the cortisol inactivating enzyme 11β-hydroxysteroiddehydrogenase type 2 (11β-HSD2) have been identified as underlying mechanisms. In addition, inhibition of CYP17A1-hydroxylase increases the risk for pseudohyperaldosteronism, as exemplified by the anti-cancer drug abiraterone. Impairment of CYP11B1 or CYP17A1-hydroxylase activity leads to increased steroidogenic activity to compensate for low cortisol levels. Subsequent accumulation of weak mineralocorticoids like 11-deoxycorticosterone (11-DOC) allows the mineralocorticoid receptor (MR) to be excessively activated. Similarly, cortisol activates the MR if its inactivation by 11β-HSD2 is inhibited, eventually leading to hypokalemia, sodium and water retention, and secondary hypertension.
Even though the underlying mechanisms and the risk are known, adrenocortical toxicity studies are not generally included in pre-clinical evaluations of newly developed drugs and no workflow has been established and agreed on to identify substances disturbing adrenal steroidogenesis. The present thesis aimed to apply and compare different approaches to determine a drug’s risk for pseudohyperaldosteronism.
The first focus of the thesis was to characterize anabolic androgenic steroid (AAS) derived adrenocortical toxicity using H295R adrenocortical carcinoma cells. AAS are widely used by athletes and the general population for their muscle enhancing properties. A common side effect is hypertension, the mechanism of which has not yet been fully elucidated. Using H295R cells and an extended version of the OECD test guideline 456, 19 AAS and 3 selective androgen receptor modulators (SARMs) were evaluated for their potential to interfere with adrenal steroidogenesis by measuring steroid levels in H295R cell culture supernatants with ultra-high pressure liquid chromatography mass spectrometry (UHPLC-MS/MS). Indeed, mesterolone, mestanolone and methenolone increased the mineralocorticoid output, potentially posing a risk for secondary hypertension. In a follow on study, extended steroid profiling based on untargeted steroidomics was performed and evaluated for its suitability to assess disturbances in adrenal steroidogenesis using five AAS. After 57 steroids have been identified with the untargeted approach, inhibitory mechanisms missed in the first AAS study based on targeted analysis could be defined. The main advantage of the untargeted approach was the possibility to group AAS according to their mode of action and to assess their steroidogenic fingerprint. Comparison of steroidogenic fingerprints by pattern matching with fingerprints of known inhibitors enables to judge on a compound’s risk for specific disruption mechanisms. It is a fast approach to prioritize follow on targeted enzyme activity experiments.
In a next step, the H295R cell model was evaluated for its suitability to predict pseudohyperaldosteronism by analyzing the inhibitory actions of the triazole antifungals posaconazole and itraconazole on adrenal CYP enzymes retrospectively. It was found that pseudohyperaldosteronism, i.e. meaning the accumulation of mineralocorticoids upstream of aldosterone, cannot be analyzed using H295R cells, since this depends on an increased stimulation of steroidogenesis due to an activation of the hypothalamic-pituitary-adrenal (HPA) axis as a result of low cortisol levels. Nevertheless, H295R cells were found to be an appropriate model to enable anticipation of pseudohyperaldosteronism if CYP11B1 and CYP17A1 are inhibited in H295R cells and if the workflow is complemented by enzyme activity assays.
Next, all systemically used triazole antifungals were evaluated for their interferences with adrenal steroidogenesis, including posaconazole, itraconazole, voriconazole, fluconazole and isavuconazole. Isavuconazole and itraconazole decreased the overall steroidogenic output, potentially increasing the risk for adrenal hyperplasia.
To find drugs with a higher specificity for the fungal CYP51 compared to human CYP enzymes, tetrazole antifungals have been developed. Their steroid fingerprints were compared to posaconazole and itraconazole to determine if similar inhibitory mechanisms can be expected. Hypotheses on enzyme inhibition were validated with UHPLC-MS/MS and enzyme activity assays. The results suggest that the risk for pseudohyperaldosteronism after treatment with the newly developed azole antifungals is low. However, reduced steroidogenic output indicate a potential risk for adrenal hyperplasia which needs further evaluation.
Another focus of the thesis was the identification of drugs so far unknown for their inhibitory action on CYP17A1 and CYP11B1, thus posing a risk for unexpected pseudohyperaldosteronism. In a first step, in silico pharmacophore-based screenings were performed. Potential hits were validated in enzyme activity assays, resulting in numerous and diverse inhibitors. Retinoic acid metabolism blocking agents and azole antifungals unselectively inhibited both CYP11B1 and CYP17A1, increasing the risk for unexpected side effects. Tipifarnib, a farnesyltransferase inhibitor was the most interesting and novel inhibitor identified in the CYP11B1 screening. CYP17A1 was strongly inhibited by mocetinostat, nolatrexed and serdemetan. The risk for pseudohyperaldosteronism after patient treatment with those inhibitors should be investigated in future studies.
In conclusion, a combination of diverse approaches is needed to characterize and identify inhibitors of adrenal steroidogenesis. If interested in the identification of inhibitors for a specific enzyme, cell-free activity assays in combination with computational screening approaches are suitable. To analyze the overall steroidogenesis, H295R cells are appropriate. Extending targeted analytic approaches by untargeted steroidomics enables grouping and fingerprinting of compounds, thus increasing the informational gain obtained from experiments with H295R cells.