Adrenocortical Inhibition Potency Assay

The in vivo adrenocortical inhibitory potencies of drugs were assessed in rats using an adrenocorticotropic hormone (ACTH)-stimulation test as previously described. ^{24 (link)} Each rat was given dexamethasone (0.2 mg/kg IV) to suppress baseline corticosterone production. Two hours later, dexamethasone was readministered and the desired dose of drug in dimethyl sulfoxide vehicle was rapidly injected through either a femoral venous catheter preimplanted by the vendor or a 24 gauge intravenous catheter placed in a tail vein. This was followed by a 1-ml normal saline flush. Immediately after the saline flush, ACTH_1–24 (25 μg/kg) was injected through the catheter followed by another normal saline flush. Fifteen minutes after ACTH_1–24 administration, a blood sample was removed from the catheter (~ 0.4 ml) for measurement of the corticosterone concentration to determine the adrenocortical response to the ACTH_1–24. The blood sample was allowed to clot at room temperature before centrifugation at 16000 g for 15 min. The corticosterone concentration in the resulting serum was determined using an Enzyme-Linked ImmunoSorbent Assay (ELISA) (Diagnostic Systems Laboratories, Webster, TX) and a 96-well plate reader (Molecular Devices). For each drug, the median adrenocortical inhibitory dose (ID50) was determined from the dose-corticosterone response relationship using a Hill equation.