Conventional and novel strategies in the treatment of adrenocortical cancer
D.E. Schteingart
University of Michigan, Ann Arbor, MI, USA
Correspondence
D.E. Schteingart University of Michigan Rm 5570 MSRB 2-Box 0678 1150 West Medical Center Drive Ann Arbor, MI 48109 USA E-mail: dschtein@umich.edu
Presented at the First International Meeting on Adrenal Disease: Basic and Clinical Aspects, Ribeirão Preto, SP, Brazil, August 31-September 2, 1999.
Received December 20, 1999 Accepted April 10, 2000
Abstract
Adrenocortical carcinoma is a highly malignant neoplasm with an incidence of two per million people per year. Several treatment strategies have resulted in temporary or partial tumor regression but very few cases have attained long survival. Surgical resection of the primary tumor and metastases is most effective. Several chemothera- peutic protocols have been employed with variable success. Mitotane (o,p’-DDD) is an adrenalytic drug effective in inducing a tumor response in 33% of patients treated. Mitotane requires metabolic transformation for therapeutic action. Tumors may vary in their ability to metabolize mitotane and the ability of tumors to transform mitotane may predict the clinical response to the drug. Preliminary data show a possible correlation between metabolic activity of neoplastic adreno- cortical tissue and response to mitotane. We have attempted to de- velop mitotane analogs with enhanced adrenalytic effect. Compared to mitotane, a di-chloro compound, the bromo-chloro and di-bromo analogs appear to have a greater effect. Future approaches to the treatment of adrenocortical carcinoma are likely to be based on blocking or reversing the biological mechanisms of tumorigenesis. Angiogenic and chemotactic mechanisms may play a role in adrenal tumor growth and inhibition of these mechanisms may result in inhibition of tumor growth. New mitotane analogs with greater adre- nalytic potential could be a promising approach to developing more effective and selective therapies for adrenal cancer. Alternative ap- proaches should attempt to suppress tumor growth by means of compounds with anti-angiogenic and anti-chemotactic activity.
Key words
· Mitotane · Acyl-chloride metabolite
· Bromo-chloro and di-bromo analogs
· Angiogenic and chemotactic chemokines
Adrenocortical carcinoma is a rare, highly malignant neoplasm which occurs with an incidence of two per million population per year. It represents 0.2% of all cases of can- cer. Several treatment strategies have resulted in temporary or partial tumor regression but very few cases have attained long survival (1). More effective therapy is needed and it is likely to come from a better understanding of tumor biology; specifically, the oncogenic
and tumorigenic processes that govern early cell mutation and the growth and dissemina- tion of an established tumor.
The difficulty in assessing the effective- ness of published treatment protocols stems from the fact that most series are limited in the number of patients studied. There is great variability in the drugs used, the stage and extent of the tumor, and the malignancy grade. In addition, there is lack of a uniform defini-
tion of response, the duration of response is unclear and multiple treatments are given in variable sequences.
Several of the larger series indicate that the most effective treatment is the surgical resection of the primary tumor and metasta- ses, with 56% of patients showing extended survival. Abdominal irradiation is associ- ated with tumor response in 15% and sys- temic chemotherapy in less than 10%. Sev- eral chemotherapeutic protocols have been employed with variable success. Combina- tions of adriamycin, vincristin, cisplatin and etoposide have been used in escalating doses with some success. Other drugs such as taxol, suramin and gossipol have been used with equivocal results.
Mitotane (o,p’-DDD) is an adrenalytic drug with selective activity on the adrenal cortex which has been found to be effective in inducing a tumor response in 33% of patients treated. The duration of response has been quite variable, ranging from 1 to 204 months. The use of mitotane as adjuvant therapy in patients with stage I and II adreno- cortical carcinoma is controversial because of lack of convincing data that the drug can prevent tumor recurrence and the significant toxicity associated with its administration.
A better understanding of the mechanism of action of mitotane could be useful in explaining its antitumor effect and in devel- oping a rationale for the design of more effective and less toxic compounds. Mito- tane belongs to the class of drugs that re- quires metabolic transformation for thera- peutic action. As a result of this transforma- tion, active metabolites are produced that cause toxicity either through covalent bind- ing to specific targets within the cells or by oxygen activation with superoxide forma- tion. The concept that mitotane requires metabolic transformation for activity stems from observations of its variable activity in different animal species. The dog adrenal, the most responsive to mitotane, is also the most capable of metabolite formation and
covalent binding. In contrast, the human ad- renal is less capable of both transformation and binding and is less responsive (2).
We proposed a pathway of mitotane me- tabolism that follows the well-known pro- cess by which chloramphenicol causes tox- icity. Mitotane is hydroxylated at the ß-car- bon and quickly transformed by dehydro- chlorination into an acyl-chloride. The acyl- chloride either covalently binds to bionucle- ophiles in the target cells or, by losing water, is transformed to the acetic acid derivative (DDA) for renal excretion. The initial hy- droxylation step is carried out in the mito- chondria and is catalyzed through a P-450 enzyme. The importance of this metabolic transformation can be tested by the introduc- tion of a methyl group at the ß-carbon of o,p’-DDD, a procedure that blocks the meta- bolic transformation to the acyl-chloride. Whereas dogs treated with mitotane show prompt suppression of cortisol secretion and increases in serum ACTH levels, treatment with the methylated analog (mitometh) lacks this effect. Similarly, while mitotane causes necrosis of the adrenal cortex, mitometh has no significant effect (3).
The pathway of metabolic transforma- tion of mitotane has been extensively stud- ied in our laboratories (4). Formation of hydroxylated derivatives can be shown by incubation of dog adrenal homogenates with radiolabeled o,p’-DDD. HPLC analysis of the homogenate after incubation shows the appearance of hydroxylated metabolites and the production of DDA. We have character- ized the adrenal enzymes involved in mito- tane metabolism. The metabolic reaction is dependent on O2 and NADPH, and is inhib- ited by 68% with a mixture of O2-CO (20:80). The metabolic transformation is inhibited by ketoconazole, but not by other specific en- zyme inhibitors such as aminoglutethimide and metyrapone, or substrates such as cho- lesterol, 11-deoxycorticosterone, 11-deoxy- cortisol, corticosterone and androstenedione. It is possible that the mitotane-metabolizing
enzyme is a novel non-steroidogenic P-450 present in the adrenal cortex and active in the metabolism of xenobiotics.
We have also investigated the cellular target to which mitotane metabolites are co- valently bound. Incubation of normal adre- nal and adrenal tumor homogenates with a radiolabeled analog of mitotane show that most of the radioactivity is associated with proteins with molecular weights of 49.5 and 11.5 kDa. The sequence and structure of these proteins have not been worked out.
Another possible mechanism mediating the adrenalytic effect of mitotane is oxida- tive damage through production of free radi- cals. We have tested the effect of tocopherol acetate on the antiproliferative activity of mitotane and shown that this activity is re- versed by the addition of this antioxidant to NCI-H295 adrenal cancer cell cultures.
Thus, metabolic transformation and free radical formation are mechanisms involved in the cytotoxicity of mitotane. Tumors vary in their ability to metabolize mitotane and the ability of tumors to transform mitotane may predict the clinical response to the drug. Preliminary data show a possible correlation between metabolic activity of neoplastic adrenocortical tissue and response to mito- tane. We have developed a tritium release assay to test the ability of adrenal tumors to metabolize mitotane (5). Tritiated mitotane is incubated with adrenal homogenates or cell suspensions. The unreacted substrate is removed and the amount of tritium released into the aqueous media is determined. The amount of tritium released correlates with the metabolic transformation into the acyl- chloride. Results from the tritium release assay correlate with data obtained using 14C- labeled compound. This assay has potential clinical applications for selecting patients who are responsive to mitotane from those who are not likely to respond.
Based on the mechanism of action of mitotane described, we have attempted to develop mitotane analogs with enhanced
adrenalytic effect. We postulated that re- placement of chlorine with more reactive halides such as bromine might increase the transformation of substrate to the acyl-chlo- ride. Two compounds, bromo-chloro and di- bromo analogs were synthesized. When these analogs were added to NCI-H295 adrenal cancer cell cultures, there was a dose-de- pendent suppression of cell growth and cor- tisol production. Compared to mitotane, the bromo-chloro and di-bromo analogs appear to have a greater effect. These effects were also shown in vivo when dogs were treated with equimolar doses of the analogs. Atro- phy of the zona fasciculata and reticularis was greater with the brominated analogs than with mitotane.
Future approaches to the treatment of adrenocortical carcinoma are likely to be based on blocking or reversing the biologi- cal mechanisms of tumorigenesis. For ex- ample, angiogenic and chemotactic mechan- isms may play a role in adrenal tumor growth. Inhibition of these mechanisms may result in inhibition of tumor growth.
We have recently described a CXC chemokine-producing adrenocortical carci- noma. The patient, a 74-year-old man, pre- sented with intermittent fever, marked leu- kocytosis and elevated acute phase reac- tants. A cell line developed from this tumor actively produced chemokines in vitro, in- cluding interleukin-8 and epithelial neutro- phil activating protein-78 (ENA-78), potent angiogenic and chemotactic chemokines. These cells were transplanted subcutane- ously into SCID mice and an animal model of this tumor was developed. The tumors were infiltrated with neutrophils and a simi- lar neutrophil infiltration was seen around the central veins in the mouse liver. Prelimi- nary studies with passive immunization against ENA-78 have shown 50% suppres- sion of tumor growth that was associated with suppression of endothelial cell and leu- kocyte markers in the tumor.
In summary, the development of new
mitotane analogs with greater adrenalytic potential could be a promising approach to developing more effective and selective therapies for adrenal cancer. Alternative ap-
proaches should attempt to suppress tumor growth by means of compounds with anti- angiogenic and anti-chemotactic activity.
References
1. Schteingart DE (1992). Treating adrenal cancer. Endocrinologist, 2: 149-157.
2. Martz F & Straw JA (1977). The in vitro biotransformations of 1-(o-chlorophenyl)- 1-(chlorophenyl)-2,2-dichloroethane (o,p’- DDD) by dog adrenal mitochondria macro- molecules. Drug Metabolism and Dispo- sition, 5: 482-486.
3. Schteingart DE, Sinsheimer JE, Counsell RE, Abrams GD, Mcclellan N, Djanegara
T, Hines J, Ruangwises N, Benitez R & Wotring LL (1993). Comparison of adre- nalytic activity of mitotane and a methyl- ated homolog on normal adrenal cortex and adrenal cortical carcinoma. Cancer Chemotherapy and Pharmacology, 31: 459-466.
4. Cai W, Counsell RE, Djanegara T, Schteingart DE, Sinsheimer JE & Wotring LL (1995). The metabolic activation and
binding of mitotane in adrenal cortex ho- mogenates. Journal of Pharmaceutical Sciences, 84: 134-138.
5. Pineiro-Sanchez ML, Vaz AND, Counsell RE, Ruyan M, Schteingart DE & Sins- heimer JE (1995). Synthesis of ß3H-mito- tane for use in a rapid assay for mitotane metabolism. Journal of Labelled Com- pounds and Radiopharmaceuticals, 36: 121-127.