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Purpose: Mitotane (o,p'-DDD), is the only adrenolytic agent available for the treatment of adrenocortical carcinoma. Previous studies have shown that mitotane covalently binds to adrenal proteins following its metabolism in adrenocortical tissue to a reactive acyl chloride intermediate. It was the objective of this study to compare the electrophoresis separation patterns of such adducts following activation of mitotane by various adrenocortical sources., Methods: With the use of a 125I-labeled analog of mitotane, 1-(2-chlorophenyl)-1-(4-iodophenyl)-2,2-dichloroethane, gel electrophoresis patterns were obtained for homogenates from bovine, canine and human adrenocortical preparations as well as from a human adrenal preparation. Western immunoblotting analysis was used to test the resulting patterns for adducts of cytochrome P-450scc and adrenodoxin., Results: The electrophoresis separations were similar for all preparations, with bands at apparent molecular weights of 49.5 and 11.5 kDa being the most pronounced. Radiolabeling of the proteins of a human adrenal cancer cell line NCI H-295 was weak, but a band at 11.5 kDa was detected. Western immunoblotting analyses indicated that the band at 49.5 kDa corresponded in molecular weight to that of adrenal cytochrome P-450scc, but the band at 11.5 kDa did not correspond to adrenodoxin., Conclusions: The similarity of the results with canine and bovine adrenal preparations to that of human material offers useful systems for studying mitotane and its analogs. This should aid in understanding the mechanism of action of mitotane and in the design of compounds for the treatment of adrenocortical carcinoma.

The adrenalytic activity of mitotane (o,p'-DDD) has made it useful in the treatment of adrenocortical carcinoma and Cushing's syndrome. In support of a study to develop mitotane analogs as more effective therapeutic agents and as a basis for understanding the toxicity of related compounds in the adrenals, the biotransformations of o,p'-DDD in adrenocortical homogenate preparations have been studied and compared with those of its m,p'- and p,p'-isomers. Aliphatic oxidation to the corresponding acetic acid derivative, o,p'-, m,p'- or p,p'-DDA, was the major transformation for all the preparations. In the comparisons of the DDD isomers, the order of both DDA formation and apparent covalent binding was o,p'- > m,p'- > p,p'-DDD. There was also evidence for alpha-hydroxylation at the benzylic carbon with subsequent loss of water to form ethylene derivatives. This was a minor pathway for o,p'-DDD, but was the major pathway for the other two isomers. Thus, while the total yields of metabolites of o,p'- and m,p'-DDD were similar and at least twice that of the p,p'-isomer, their distribution of metabolites differed significantly. The effects of the three isomers on cell growth and cortisol production with the human adrenocortical carcinoma cell line, NCI H-295, followed the same order as their DDA formation and tissue binding. It is proposed that hydroxylation by the adrenal cortex at the beta-carbon leads to an adrenalytic effect, whereas hydroxylation at the alpha-carbon would represent an alternate deactivation pathway.

Mitotane [1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane, o,p'-DDD] is an adrenocorticolytic agent of value in the treatment of adrenocortical carcinoma and Cushing's syndrome. In support of a program to develop agents superior to mitotane, it is the purpose of this study to explore the relationship of the metabolism of mitotane to its binding to adrenal cortex tissue from several sources. The objective was to detect the mitotane moiety responsible for its covalent binding in various test systems. Studies were conducted with an 125l-labeled analog of mitotane, 1-(2-chlorophenyl)-1-(4-iodophenyl)-2,2-dichloroethane, prior to a comparison to results with lower specific activity [14C]mitotane. With dog adrenal cortical whole homogenates, the majority of covalent binding was to proteins with an additional one-sixth of the total bound radioactivity associated with a phospholipid fraction. No radioactivity was associated with DNA. The rank order of species in regard to metabolism and protein binding was bovine > dog > rat adrenal homogenates > human normal adrenal or tumor homogenates. The percentage of radioactivity recovered from the hydrolysates of those fractions was uniformly high. In addition, the only metabolite present in the hydrolysates corresponded to 1-(2-chlorophenyl)-1-(4-iodophenyl)acetic acid from the iodo analog of o,p'-DDD and the corresponding o,p'-dichlorodiphenylacetic acid (o,p'-DDA) from o,p'-DDD. Our results are consistent with an acyl chloride being the reactive intermediate formed from the dichloromethyl moiety of mitotane, which leads to both DDA metabolite formation and binding to adrenal cortical bionucleophiles.

Mitotane is an important adrenalytic drug for the treatment of adrenal cancer whose use is limited by toxicity. Reports from another laboratory indicated that a methylated homolog of Mitotane (Mitometh) tested in guinea pigs possessed comparable adrenalytic activity but was less toxic than Mitotane. This observation prompted us to undertake a comparative study of these two drugs on the basis that Mitometh may be a superior agent for the treatment of adrenal cancer. Preliminary studies in guinea pigs failed to show a significant adrenalytic effect for either Mitotane or Mitometh. Thus, we extended the study to 13 mongrel dogs weighing 12-15 kg that were treated daily with Mitometh or Mitotane (50-100 mg/kg) for 6 or 12 days. Cortisol decreased to undetectable levels and adrenocorticotropic hormone (ACTH) rose to 10 times the baseline levels within 72 h in Mitotane-treated animals. Despite the achievement of similar drug levels, Mitometh treatment in dogs failed to suppress cortisol or increase ACTH. To determine whether these differences were due to differences in bioavailability, we measured the relative concentration of Mitotane and Mitometh in homogenates of adrenal cortex obtained from Mitotane- and Mitometh-treated dogs. The adrenal concentration of Mitometh determined in Mitometh-treated dogs was 5 times higher than the concentration of Mitotane measured in Mitotane-treated animals. Whereas the adrenal glands of Mitotane-treated dogs showed hemorrhage and necrosis, the Mitometh-treated animals showed no adrenal damage. Despite the lack of adrenalytic activity, Mitometh maintained its toxicity as demonstrated by microscopic evidence of hepatic necrosis and an increase in hepatic enzymes. The adrenalytic effects of both agents was also studied in vitro using a human functioning adrenal cortical carcinoma cell line, NCI-H295. Whereas Mitotane strongly suppressed cell growth, Mitometh had a weaker effect. We conclude that Mitometh is not likely to be effective in the therapy of adrenal cancer. Moreover, the results of this study are supportive of the view that metabolic transformation of Mitotane is in some way linked to its adrenalytic action.

Dexamethasone suppression (DS) adrenal cortical scintigraphy has been shown to be useful in the detection of the adrenal pathology in women with hyperandrogenism. However, a relationship between adrenal cortical uptake of I-131 6 beta-iodomethylnorcholesterol (NP-59) and the level of adrenal androgen secretion has not been established. A retrospective analysis of DS adrenal scintiscans has been performed on 39 women with hirsutism and hyperandrogenism. In 14 patients with normal patterns of imaging, in vivo adrenal gland iodocholesterol uptake, calculated using a semi-operator-independent-computer algorithm, did not correlate with the excretion of urinary 17-ketosteroids (17-KS). In contrast, in 20 patients demonstrating abnormal bilateral early imaging patterns, adrenal gland NP-59 uptake correlated significantly with the level of urinary 17-KS excretion (r = 0.65, P less than 0.05). To date seven of these 20 patients have had confirmatory procedures documenting the adrenal glands as contributing sites of androgen secretion. A similar correlation with urinary 17-KS excretion was seen in five other patients with unilateral imaging patterns (r = 0.94, P less than 0.005), due to androgen-secreting adrenal cortical adenomas. No correlation between adrenal NP-59 uptake and plasma testosterone or dehydroepiandrosterone sulphate levels was observed in any of the groups. Thus, adrenal gland uptake of NP-59 under DS reflects a measure of androgen secretion in women with androgen excess.

The adrenal gland uptake of 131I-6-beta-iodomethyl-19-norcholesterol (NP-59) was calculated using a semioperator-independent computer algorithm in 17 patients with Cushing's syndrome (CS). Twelve had ACTH-dependent and 5 had ACTH-independent CS. The mean adrenal gland uptake (percentage of administered dose) of NP-59 was 0.74 +/- 0.18% (range, 0.21-2.02%) in CS and was significantly higher than that of normal subjects (0.33 +/- 0.02%). Patients with the ectopic ACTH syndrome and ACTH-independent CS manifested the highest uptakes (mean, 1.18 +/- 0.08%; range, 0.74-2.02%). In the patients with ACTH-dependent CS, a significant correlation was observed between 24-h urinary free cortisol excretion and NP-59 adrenal gland uptake (r = 0.97, P less than 0.001). No relationship was seen between NP-59 uptake and urinary free cortisol in ACTH-independent CS or between NP-59 uptake and urinary 17-hydroxycorticosteroids, 17-ketosteroids, cortisol secretion rate, plasma cortisol, or ACTH levels in either ACTH-dependent or ACTH-independent CS. We conclude that in addition to localizing the sites(s) of adrenocortical hypersecretion in CS, the level of NP-59 adrenal uptake is a reflection of cortisol excretion in ACTH-dependent disease and may be utilized as another parameter to quantitate adrenal disease activity in CS.

Study Objective: To assess the efficacy of 131I-6-beta-iodomethylnorcholesterol scintigraphy in the adrenocorticotropic hormone-independent Cushing syndrome and to compare this with computed tomography., Design: Retrospective analysis of case series from 1977 to 1987., Setting: Referral to the Division of Nuclear Medicine at a tertiary-care university medical center., Patients: Twenty-four patients with a pathologically-confirmed diagnosis of the adrenocorticotropic hormone-independent Cushing syndrome had 131I-6-beta-iodomethylnorcholesterol scintigraphy and, in most cases, computed tomography., Measurements and Main Results: Using 131I-6-beta-iodomethylnorcholesterol scintigraphy, adenomas were accurately seen as focal, unilateral tracer uptake in 14 of 14 patients. In carcinoma, the classic scintigraphic pattern of bilateral nonvisualization was observed in 3 of 4 patients, with ipsilateral uptake of tracer in 1 patient with a histologically well-differentiated malignancy. Computed tomography done during the same interval depicted abnormal adrenals in all cases of adenoma and carcinoma. In cortical nodular hyperplasia, however, computed tomography identified abnormal pairs of adrenals in only one of four cases studied, whereas scintigraphy showed typical patterns of bilateral increased uptake in all of the cases., Conclusion: 131I-6-beta-iodomethylnorcholesterol scintigraphy accurately shows the location and nature of adrenal dysfunction in the adrenocorticotropic hormone-independent Cushing syndrome and may be particularly useful in identifying the bilateral adrenal involvement in cortical nodular hyperplasia.