Mitotane Therapy in Adrenocortical Cancer Induces CYP3A4 and Inhibits 5x-Reductase, Explaining the Need for Personalized Glucocorticoid and Androgen Replacement
Vasileios Chortis,* Angela E. Taylor,* Petra Schneider, Jeremy W. Tomlinson, Beverly A. Hughes, Donna M. O’Neil, Rossella Libé, Bruno Allolio, Xavier Bertagna, Jérôme Bertherat, Felix Beuschlein, Martin Fassnacht, Niki Karavitaki, Massimo Mannelli, Franco Mantero, Giuseppe Opocher, Emilio Porfiri, Marcus Quinkler, Mark Sherlock, Massimo Terzolo, Peter Nightingale, Cedric H. L. Shackleton, Paul M. Stewart, Stefanie Hahner, and Wiebke Arlt1
Context: Mitotane [1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane] is the first-line treat- ment for metastatic adrenocortical carcinoma (ACC) and is also regularly used in the adjuvant setting after presumed complete removal of the primary tumor. Mitotane is considered an adrenolytic sub- stance, but there is limited information on distinct effects on steroidogenesis. However, adrenal in- sufficiency and male hypogonadism are widely recognized side effects of mitotane treatment.
Objective: Our objective was to define the impact of mitotane treatment on in vivo steroidogenesis in patients with ACC.
Setting and Design: At seven European specialist referral centers for adrenal tumors, we analyzed 24-h urine samples (n = 127) collected from patients with ACC before and during mitotane therapy in the adjuvant setting (n = 23) or for metastatic ACC (n = 104). Urinary steroid metabolite excretion was profiled by gas chromatography/mass spectrometry in comparison with healthy controls (n = 88).
Results: We found a sharp increase in the excretion of 60-hydroxycortisol over cortisol (P < 0.001), indicative of a strong induction of the major drug-metabolizing enzyme cytochrome P450 3A4. The contribution of 6ß-hydroxycortisol to total glucocorticoid metabolites increased from 2% (median, interquartile range 1-4%) to 56% (39-71%) during mitotane treatment. Furthermore, we docu- mented strong inhibition of systemic 5a-reductase activity, indicated by a significant decrease in 5a-reduced steroids, including 5a-tetrahydrocortisol, 5a-tetrahydrocorticosterone, and andros- terone (all P < 0.001). The degree of inhibition was similar to that in patients with inactivating 5a-reductase type 2 mutations (n = 23) and patients receiving finasteride (n = 5), but cluster analysis of steroid data revealed a pattern of inhibition distinct from these two groups. Longitu- dinal data showed rapid onset and long-lasting duration of the observed effects.
Conclusions: Cytochrome P450 3A4 induction by mitotane results in rapid inactivation of more than 50% of administered hydrocortisone, explaining the need for doubling hydrocortisone replace- ment in mitotane-treated patients. Strong inhibition of 5a-reductase activity is in line with the clinical observation of relative inefficiency of testosterone replacement in mitotane-treated men, calling for replacement by 5x-reduced androgens. (J Clin Endocrinol Metab 98: 161-171, 2013)
* V.C. and A.E.T. are equal first authors.
t Author affiliations are shown at the bottom of the next page. Abbreviations: ACC, Adrenocortical carcinoma; An/Et, ratio of androsterone to etio- cholanolone; CI, confidence interval; CYP, cytochrome P450; GC, gas chromatography; LDA, linear discriminant analysis; MS, mass spectrometry; 6BOHF/F, ratio of 6ß-hydroxy- cortisol to cortisol; PCA, principal component analysis; Q1, quartile 1; 5@-THB/THB, ratio of 5a-tetrahydrocorticosterone/tetrahydrocorticosterone; 5@-THF/THF, ratio of 5@-tetra- hydrocortisol to tetrahydrocortisol.
A drenocortical carcinoma (ACC) is a rare cancer, with an incidence of one to two cases per million per year and a poor prognosis, mostly due to a high risk of recurrence and limited therapeutic options (1). Mi- totane [1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-di- chloroethane (o,p’-DDD)], an analog of the insecticide dichlorodiphenyltrichloroethane (DDT), has been used in the treatment of ACC since 1959 (2). Mitotane alone or in combination with cytotoxic chemotherapy is now established as the first-line treatment for metastatic ACC (3-8) and is also widely used as adjuvant therapy in patients with apparently complete surgical removal of the primary tumor, especially if considered at high risk of recurrence (9).
Despite the widespread use of mitotane in adrenal cancer, there is limited knowledge regarding the mech- anisms underlying its antitumor activity, usually de- scribed as adrenolytic, i.e. a direct cytotoxic effect on the adrenal cortex (3, 10-12). There is also a paucity of information on distinct effects of mitotane on steroid- ogenesis, although it has been noted early on as an ef- ficient treatment for Cushing’s syndrome (13-16), and in patients with normal adrenal function, mitotane ther- apy invariably results in adrenal insufficiency. There is in vivo evidence of enhanced production of cortisol- binding globulin and SHBG in mitotane-treated pa- tients (17, 18). Notably, glucocorticoid replacement has to be administered in higher doses than usual in the general context of adrenal insufficiency to prevent ad- renal crisis (3, 19). Mitotane-induced hypogonadism is frequently observed in male patients (18), but testos- terone replacement often lacks clinical efficacy.
We have recently shown that urine steroid metabo- lomics, i.e. the combination of steroid profiling by gas chromatography (GC)/mass spectrometry (MS) and computational data analysis, is a highly promising di- agnostic tool for the detection of adrenocortical malig- nancy (20). Here we investigated the effects of mitotane on in vivo steroid production employing urinary steroid metabolomics for the analysis of 24-h urine samples from patients with adrenal cancer receiving mitotane for adjuvant treatment or metastatic disease.
Subjects and Methods
Subjects
The 24-h urine samples from ACC patients were collected between 2006 and 2010 in seven specialist endocrine referral centers participating in the European Network for the Study of Adrenal Tumors (ENSAT; www.ensat.org), with approval of local ethical review boards and after obtaining written informed patient consent. We included 24-h urines from 100 patients (53 women, 47 men; median age 52, range 16-80 yr) with histo- logically confirmed ACC who provided a total of 127 samples including 46 paired samples. Samples were collected before (ADJ, n = 12) and during (ADJ+M, n = 11) adjuvant mitotane therapy or before (MET, n = 57) and during (MET+M, n = 47) mitotane treatment for metastatic ACC. Samples during mito- tane treatment were collected 3-4 months after initiation of ther- apy, i.e. at a time when therapeutic-range plasma mitotane levels (14-20 mg/liter) (21) generally had been achieved. None of the patients on adjuvant therapy had documented recurrence during this initial treatment period, and there were no major changes in tumor burden as documented by imaging in the metastatic group patients. Plasma mitotane levels were available for 50 of the 58 patients on mitotane, all of them measured by HPLC (Lysosafe, Paris, France). Exclusion criteria included pregnancy and expo- sure to drugs known to induce expression and activity of hepatic cytochrome P450 (CYP) enzymes or to alter steroid secretion in any way, with the exception of glucocorticoid replacement ther- apy, which was routinely commenced in all mitotane-treated patients.
For comparison, we employed 24-h urine samples of 88 healthy controls (62 females, 26 males, age range 18-60 yr). In addition, for the assessment of 5a-reductase activity, we also compared the results with 24-h urine samples from patients with inactivating mutations in SRD5A2 encoding 5a-reductase type 2 (n = 23) and patients treated with the 5a-reductase type 2 inhibitor finasteride (n = 5).
GC/MS urinary steroid metabolome analysis
Measurement of 24-h urinary steroid metabolite excretion was carried out by GC/MS as previously described (20). In sum- mary, free and conjugated steroids were extracted from urine by solid-phase extraction. Steroid conjugates were enzymatically hydrolyzed, reextracted, and chemically derivatized to form methyloxime trimethyl silyl ethers. GC/MS analysis of the urine samples was carried out on an Agilent (Santa Clara, CA) 5973 instrument operating in selected-ion-monitoring (SIM) mode. This achieved sensitive and specific detection and quantification of 32 selected steroid metabolites chosen to include important representatives of steroid groups such as androgen metabolites, glucocorticoid metabolites, mineralocorticoid metabolites, and
Centre for Endocrinology, Diabetes, and Metabolism (V.C., A.E.T., P.S., J.W.T., B.A.H., D.M.O’N., M.S., C.H.L.S., P.M.S., W.A.), School of Clinical and Experimental Medicine, and School of Cancer Sciences (E.P.), University of Birmingham, Birmingham B15 2TT, United Kingdom; Department of Endocrinology (R.L., J.B., X.B.), Institut National du Cancer Cortico-Medullo Tumeurs Endocrines, Cochin Hospital, Institut Cochin, Institut National de la Santé et de la Recherche Médicale Unité 1016, René Descartes University, Paris, F-75006 France; Endocrine and Diabetes Unit (S.H., B.A., M.F.), Department of Medicine I, University Hospital, University of Würzburg, D-97080 Würzburg, Germany; Endocrine Research Unit (F.B., M.F.), Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, D-80336 Munich, Germany; Department of Endocrinology (N.K.), John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom; Endocrinology Unit (M.M.), Department of Clinical Pathophysiology, University of Florence and Istituto Toscano Tumori, I-50121 Florence, Italy; Familial Cancer Clinic (G.O.) and Division of Endocrinology (F.M.), Veneto Institute of Oncology Instituto di Ricovero e Cura a Carattere Scientifico and Department of Medical and Surgical Sciences, University of Padova, I-35100 Padova, Italy; Clinical Endocrinology (M.Q.), Charité Campus Mitte, Charité University Medicine Berlin, D-10117 Berlin, Germany; Department of Endocrinology (M.S.), Tallaght Hospital, and Department of Medicine, Trinity College Dublin, Dublin 2, Ireland; Department of Clinical and Biological Sciences (M.T.), Internal Medicine I, University of Turin, I-10124 Turin, Italy; and Wellcome Trust Clinical Research Facility (P.N.), University Hospital Birmingham National Health Service Foundation Trust, Birmingham B15 2TH, United Kingdom
3B-hydroxy-A (5) steroid metabolites (for details of the steroid metabolite profile see Ref. 20).
After analysis of the entire profile, we calculated substrate metabolite to product metabolite ratios to assess the effects of mitotane on the in vivo net activity of distinct steroidogenic en- zymes. This included 5a-reductase indicated by the ratio of 5a- tetrahydrocortisol to tetrahydrocortisol (5a-THF/THF), ratio of androsterone to etiocholanolone (An/Et), and ratio of 5@-tetra- hydrocorticosterone/tetrahydrocorticosterone (5a-THB/THB). As a measure of the activity of the major drug-metabolizing en- zyme CYP3A4, we calculated the ratio of 6ß-hydroxycortisol to cortisol (6BOHF/F) (22). Total steroid output was calculated as the sum of all quantified steroid metabolites with the exception of glucocorticoid metabolites because these also reflected exog- enously administered glucocorticoid replacement. Total when used in this paper relates to the targeted compounds measured, which are dominant metabolites of hormonal steroids and their precursors. It does not include a multitude of minor metabolites.
Statistical analysis
Diagnostic ratios were presented as median and interquartile ranges [quartile 1 (Q1)-Q3]. In a first analysis, we considered all samples to be independent and employed nonparametric Kruskal-Wallis test and Dunn’s post hoc test to detect significant differences of individual ratios among the treatment groups. To take the paired nature of a subset of samples into account, we provided a separate analysis of the data employing Wilcoxon signed rank test. These analyses were carried employing Sigma- Plot (Systat Software Inc., Chicago, IL).
Furthermore, we analyzed the influence of mitotane on 5a- reductase activity by performing multivariate analyses of the ratios reflecting 5a-reductase activity, An/Et, 5a-THF/THF, and 5a-THB/THB. We performed principal component analysis (PCA) and linear discriminant analysis (LDA) (23) to generate two-dimensional representations of the data. Prior to analysis, the values of the ratios were log-transformed and normalized to zero mean and unit variance. The generated scatter plots allowed identification of clusters of similar ratio profiles. These analyses were done using the software MATLAB (Mathwork Inc., Natick, MA).
Finally, we analyzed the association between plasma mito- tane levels and the values of the steroid ratios indicative of 5x- reductase and CYP3A4 activities by computing Spearman’s rank correlation coefficient, thereby accounting for the lack of normal distribution of the data. This analysis was carried out employing MATLAB.
Results
Mitotane down-regulates overall steroidogenesis
First, we analyzed total steroid excretion to assess whether mitotane has an impact on the initial steps of steroidogenesis, namely CYP11A1 activity converting cholesterol to pregnenolone. For this analysis, we dis- regarded active glucocorticoid metabolites because the mitotane-treated patients invariably received hydrocor- tisone replacement therapy, which prevented a compre- hensive assessment of endogenous glucocorticoid pro-
duction. Comparing the remainder of total steroid excretion, we found that mitotane led to a significant down-regulation of overall steroidogenesis in meta- static ACC patients, as documented by a significant decrease in the sum of total androgen and mineralocor- ticoid metabolites (Fig. 1A and Table 1). This down- regulation was significant for the larger group of met- astatic ACC patients, decreasing both androgen and mineralocorticoid excretion to the level found in healthy controls, but failed to reach significance for the smaller adjuvant therapy group (Fig. 1A and Table 1).
Of note, we found that the excretion of the 11-deoxy- cortisol metabolite tetrahydro-11-deoxycortisol did not differ when comparing steroid excretion before and after the initiation of mitotane therapy (Fig. 1B and Table 1), indicating that mitotane had no effect on 11ß-hydroxylase activity, which converts 11-deoxycortisol to cortisol.
Mitotane induces CYP3A4 activity and glucocorticoid inactivation
After the initiation of mitotane treatment, 6BOHF/F showed a significant increase in both patients with meta- static disease and patients receiving adjuvant therapy (Fig. 1, C and D, and Table 1 and Supplemental Table 1, pub- lished on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org), which was due to large increases in 6ß-OHF excretion, indicative of a highly en- hanced rate of inactivation of cortisol to 6BOHF, a con- version predominantly catalyzed by CYP3A4 (24-26). Before mitotane treatment, 6BOHF represented only 1.3% (ADJ; Q1-Q3 1.0-2.6%) and 1.8% (MET; Q1-Q3 0.8-4.2%), respectively, of total measured glucocorticoid metabolite excretion. By contrast, during mitotane treat- ment, 63.8% (ADJ+M; Q1-Q3 48.9-70.2%) and 52.5% (MET+M; Q1-Q3 38.8-69.1%), respectively, of mea- sured glucocorticoids were excreted as 6BOHF (Fig. 1E), suggesting rapid inactivation of exogenously adminis- tered hydrocortisone in the mitotane-treated patients.
To exclude a significant contribution of the concurrent hydrocortisone replacement on the induction of CYP3A4 observed in the mitotane-treated patients, we also studied the percentage of 6BOHF as part of total glucocorticoid excretion in patients with adrenal insufficiency, specifi- cally 30 patients on a regular-dose hydrocortisone re- placement (10-30 mg/d) and 10 patients who received 400 mg hydrocortisone during the 24-h period of urine collection. Results revealed that 6BOHF on regular hy- drocortisone dose did not differ from healthy controls, whereas high-dose hydrocortisone slightly increased the median percentage of 6BOHF to just under 5% (Fig. 1E), confirming an only very minor contribution of hydrocor- tisone to the observed effect.
A
Androgens + Mineralocorticoids
B
Tetrahydro-11-deoxycortisol
1000000
100000
100000
10000
ug/24hr
µg/24hr
1000
10000
100
1000
10
100
1
Controls
MET
MET+M
ADJ
ADJ +M
Controls
MET
MET+M
ADJ
ADJ +M
C
CYP3A4 activity (lin)
D
CYP3A4 activity (log)
40
100
30
6BOHF/F
6BOHF/F
10
20
1
10
0
0.1
Controls
MET
MET+M
ADJ
ADJ +M
Controls
MET
MET+M
ADJ
ADJ +M
E
MET
MET+M
ADJ
ADJ+M
Controls
AI HC 10-30mg/day
AI HC 400mg/day
6BOH F ☐ Other glucocorticoids
GC/MS analysis of urine also showed a major increase in the excretion of normally minor metabolites formed through CYP3A4 activity but not selected for quantitation in this study, notably 6a- and 18-hydroxylated metabo- lites of tetrahydrocortisone and the cortolones. We did not find an effect of mitotane on the steroid ratios reflective of 11ß-hydroxysteroid dehydrogenase type 1 or 2 activity.
Mitotane inhibits 5a-reductase activity and androgen activation
Introduction of mitotane therapy resulted in a highly significant decrease of several steroid metabolite ratios reflective of systemic 5a-reductase activity (Fig. 2 and Ta- ble 1 and Supplemental Table 1). The degree of inhibition of 5a-reductase activity appeared to be similar to that
| Controls (n = 88) | MET (n = 57) | MET+M (n = 47) | ADJ (n = 12) | ADJ+M (n = 11) | |
|---|---|---|---|---|---|
| Total steroid excretion (steroid metabolites 1-19 without active glucocorticoid metabolites 20-32) | |||||
| Median | 5,579 | 22,468 | 6,494 | 2,908 | 1,137 |
| Q1-Q3 | 3,702-7,982 | 9,470-88,311 | 1,660-22,130 | 1,849-6,472 | 548-22,208 |
| P1 | <0.0001 | 1.0 | 0.60 | <0.01 | |
| P2 | <0.0001 | 0.97 | |||
| Tetrahydro-11-deoxycortisol (THS)-11-deoxycortisol metabolite | |||||
| Median | 47 | 402 | 356 | 79 | 90 |
| Q1-Q3 | 33-63 | 88-4539 | 108-3515 | 60-107 | 41-184 |
| P1 | <0.0001 | <0.0001 | 0.14 | 0.74 | |
| P2 | 1.0 | 1.0 | |||
| 60-OHF/F - CYP3A4 activity | |||||
| Median | 2.38 | 1.53 | 21.7 | 2.34 | 29.1 |
| Q1-Q3 | 1.83-3.11 | 0.53-2.17 | 13.0-26.4 | 1.90-2.93 | 28.3-33.8 |
| P1 | 0.77 | <0.0001 | 1.00 | <0.0001 | |
| P2 | <0.0001 | <0.0001 | |||
| 5a-THF/THF - 5a-reductase activity | |||||
| Median | 0.70 | 0.58 | 0.02 | 0.34 | 0.02 |
| Q1-Q3 | 0.52-0.95 | 0.18-4.54 | 0.01-0.05 | 0.26-0.93 | 0.01-0.03 |
| P1 | 1.00 | <0.0001 | 1.00 | <0.0001 | |
| P2 | <0.0001 | <0.01 | |||
| An/Et - 5a-reductase activity | |||||
| Median | 0.88 | 0.45 | 0.20 | 0.50 | 0.20 |
| Q1-Q3 | 0.67-1.21 | 0.22-0.79 | 0.13-0.34 | 0.41-0.82 | 0.14-0.53 |
| P1 | <0.0001 | <0.0001 | 0.25 | <0.0001 | |
| P2 | <0.01 | 0.53 | |||
| 5a-THB/THB - 5a-reductase activity | |||||
| Median | 2.01 | 1.27 | 0.01 | 1.52 | 0.01 |
| Q1-Q3 | 1.48-2.66 | 0.49-3.22 | 0.00-0.33 | 0.95-2.36 | 0.00-0.03 |
| P1 | 0.20 | <0.0001 | 1.00 | <0.0001 | |
| P2 | <0.0001 | <0.01 |
For the calculation of differences in total steroid excretion, glucocorticoids were excluded because mitotane therapy requires concurrent hydrocortisone replacement, which obscures any differences in endogenous glucocorticoid secretion. Results are presented as median and the range of the 25-75th percentile (Q1-Q3). Statistical analysis was performed employing Kruskal-Wallis nonparametric testing and Dunn’s post hoc test. P1, Comparison vs. controls; P2, comparison of MET vs. MET+M and ADJ vs. ADJ+M, respectively. ADJ, Before adjuvant mitotane therapy; ADJ+M, during adjuvant mitotane therapy; MET, before mitotane treatment for metastatic ACC; MET+M, during mitotane treatment for metastatic ACC.
observed in patients receiving treatment with the estab- lished 5a-reductase type 2 inhibitor finasteride (n = 5) and patients with inactivating 5a-reductase type 2 (SRD5A2) mutations (n = 25) (Fig. 2 and Table 2). However, of note, the 5a-THB/THB ratio was more significantly inhibited by mitotane than observed in finasteride-treated or SRD5A2 mutant patients (Fig. 2 and Table 2).
To examine the pattern of inhibition of 5a-reductase in further detail, we carried out cluster analysis employing both LDA and PCA; for this analysis, we considered all three ratios reflective of 5a-reductase activity and selected the patients receiving mitotane in the adjuvant setting to exclude any influence of tumor-related steroid produc- tion. Visualization of the data convincingly demonstrated strongly overlapping clustering of finasteride-treated pa- tients and SRD5A2 mutant patients, who both have selec-
tive loss of 5a-reductase type 2 activity (Fig. 3A), indicative of similar ratio profiles in these two groups. By contrast, patients receiving mitotane are clearly separate in a second cluster (Fig. 3A). These findings were confirmed by an inde- pendent cluster analysis employing LDA (Fig. 3B).
Longitudinal studies
We analyzed the longitudinal course of the steroid ra- tios indicative of CYP3A4 activity (Fig. 4A) and 5a-re- ductase activity (Fig. 4, B and C) in five patients receiving adjuvant mitotane therapy for adrenocortical cancer. Re- sults demonstrate a rapid onset of the effects of mitotane on the enzymatic activities of CYP3A4 and 5a-reductase, with the full extent of the effect already documented shortly after initiation of mitotane treatment (Fig. 4, A-C).
5a-reductase activity (lin)
5a-reductase activity (log)
12
T
100
5aTHF/THF
5aTHF/THF
10-
4.
(log)
1.
2
0.1
0.01
**
0
0.001
Controls MET MET+M ADJ ADJ +M FIN 5AR2
Controls MET MET+M ADJ ADJ +M FIN 5AR2
1.6
10
1.2
An/Etio
An/Etio
1
(log)
**
0.8
**
0.1
0.4
0
Controls MET MET+M ADJ ADJ +M FIN 5AR2
0.01
Controls MET MET+M ADJ ADJ +M FIN 5AR2
5
10
5aTHB/THB
4
5aTHB/THB
1
**
3
(log)
0.1
2
1
0.01
0
Controls MET MET+M ADJ ADJ +M FIN 5AR2
0.001
Controls MET MET+M ADJ ADJ +M FIN 5AR2
We had the opportunity to document the diagnostic steroid ratios in one patient throughout 2 yr of adjuvant mitotane treatment followed by 2 yr of posttreatment ob- servation (Fig. 4D). Plasma mitotane levels oscillated within the suggested therapeutic range (14-20 mg/liter) throughout the treatment period and only became unde- tectable 1 yr after the last administration of mitotane. Concurrently, the steroid ratios indicative of CYP3A4 and 5a-reductase activity started to recover but had not re- turned to pretreatment levels even 2 yr after the end of treatment (Fig. 4D), suggestive of long-lasting effects.
Plasma mitotane levels and observed effects on steroidogenesis
We analyzed the correlation between circulating plasma mitotane levels and the severity of CYP3A4 in- duction and 5a-reductase inhibition, respectively (Supple- mental Fig. 1). This revealed a significant correlation be- tween plasma mitotane levels and the induction of
CYP3A4 [r = 0.328, 95% confidence interval (CI) = 0.055-0.556, P = 0.02] but not with the steroid ratios indicative of 5a-reductase activity (r = - 0.053, 95% CI = -0.327-0.229, P = 0.71 for 5@THF/THF; r = 0.106, 95% CI = - 0.178-0.373, P = 0.46 for An/Et) (Supple- mental Fig. 1). Significant effects on the enzymatic activ- ities were already observed at very low plasma mitotane levels and clearly below the suggested therapeutic range of 14-20 mg/liter (21) (Supplemental Fig. 1). These findings are in line with the above-described observation that urine metabolite excretion showed the full effects as early as 1-2 months after initiation of mitotane treatment (Fig. 4, A-C), when in most instances, plasma mitotane levels would not have reached the therapeutic range.
Discussion
This study documented comprehensive in vivo evidence for a strong inhibition of 5a-reductase activities and a
| Steroid ratio reflective of 5a-reductase activity | MET+M (n = 49) | ADJ+M (n = 11) | FIN (n = 5) | SRD5A2 (n = 25) |
|---|---|---|---|---|
| 5a-THF/THF | ||||
| Median | 0.02 | 0.02 | 0.02 | 0.02 |
| Q1-Q3 | 0.01-0.05 | 0.01-0.03 | 0.01-0.02 | 0.02-0.03 |
| P1 | 0.25 | 1.0 | ||
| P2 | 1.0 | 0.52 | ||
| An/Et | ||||
| Median | 0.20 | 0.20 | 0.16 | 0.23 |
| Q1-Q3 | 0.13-0.34 | 0.14-0.53 | 0.10-0.21 | 0.16-0.267 |
| P1 | 0.79 | 1.0 | ||
| P2 | 0.75 | 1.0 | ||
| 5a-THB/THB | ||||
| Median | 0.01 | 0.01 | 0.15 | 0.14 |
| Q1-Q3 | 0.00-0.33 | 0.00-0.03 | 0.12-0.51 | 0.09-0.50 |
| P1 | 0.35 | <0.01 | ||
| P2 | 0.05 | <0.01 |
Results are presented as median and the range of the 25-75th percentile (Q1-Q3). Statistical analysis was performed employing Kruskal-Wallis nonparametric testing and Dunn’s post hoc test. P1, Comparisons vs. MET+M; P2, comparisons vs. ADJ+M. ADJ, Before adjuvant mitotane therapy; ADJ+M, during adjuvant mitotane therapy; MET, before mitotane treatment for metastatic ACC; MET+M, during mitotane treatment for metastatic ACC.
significant induction of hepatic CYP3A4/5 activities in mitotane-treated patients, with an obvious and impor- tant impact on the requirements for glucocorticoid and androgen replacement during mitotane therapy.
Evidence for alterations of cortisol metabolism and a link to hepatic enzyme activity was documented shortly after the introduction of mitotane for the treatment of adrenal cancer in 1959 (2). In 1964, two groups re- ported on altered cortisol metabolism resulting in in- creased urinary excretion of 6BOHF in guinea pig (27) and humans (28), respectively. Following up on these reports, two groups documented increased metabolism of pentobarbital (29) and hexobarbital and cortisol (30) by mitotane, postulating the induction of microsomal drug-metabolizing liver enzymes as the underlying cause. Work in the late 1980s demonstrated that the major drug-metabolizing enzyme CYP3A4 and to a lesser degree also CYP3A5 were the enzymes responsi- ble for 60-hydroxylation in liver and kidney (24-26). Subsequently, the urinary 6BOHF/F has been widely implemented as a relative measure of CYP3A4/5 in vivo activity and several studies demonstrated a 4- to 7-fold increase in 6BOHF excretion in patients treated with rifampicin (25) or anticonvulsants (31). This study an- alyzing 24-h urine samples from 127 patients collected before and during mitotane treatment demonstrated a 10- to 15-fold increase in 6BOHF/F excretion, estab- lishing mitotane as one of the strongest inducers of CYP3A4 activity. This convincingly corroborates a re-
cent report on accelerated midazolam metabolism in four mitotane-treated patients suggestive of induction of CYP3A4 (and CYP3A5) activity (32).
Early reports on hepatic enzyme induction by mitotane and also the invariable finding of highly increased cortisol- binding globulin levels during mitotane treatment (17) suggested an increased dose requirement for glucocorti- coid replacement in mitotane-induced adrenal insuffi- ciency. However, this has been widely recognized only in recent years, after a number of reports on adrenal crisis during mitotane treatment covered only with standard glucocorticoid replacement doses (19, 33). Consequently, the perceived rate of reported gastrointestinal toxicity dur- ing mitotane treatment has declined over recent years, be- cause many of these signs and symptoms may have been reflective of incipient adrenal crisis. However, currently, there is no uniformly agreed dose regimen for glucocor- ticoid replacement during mitotane therapy and reported doses have varied widely (17, 18), with lower doses often reported as associated with a high incidence of vomiting and severe fatigue (18, 34). Our findings provide for the first time a reliable quantification of 6ß-hydroxylation by mitotane, documenting the rapid excretion of 55-65% of glucocorticoids in the form of 6BOHF.
Of note, CYP3A4/5 are responsible for not only the con- version of cortisol to 6BOHF but also for the 6ß-hydroxy- lation of testosterone. Hypogonadism in mitotane-treated men initially manifests with a decreased free androgen index due to significantly increased SHBG levels that cannot be
A
Principal component analysis (PCA)
ADJ+M
FIN
5AR2
B Linear-discriminant analysis (LDA)
ADJ+M
FIN
5AR2
compensated for by up-regulated testosterone production as documented by increased total testosterone and LH levels (35). With time, gonadal testosterone production ex- hausts itself and circulating testosterone levels drop, ac- companied by clinical manifestations of low testosterone including erectile dysfunction. However, testosterone re- placement is often clinically ineffective and is complicated by an increased rate of gynecomastia (18).
This study has yielded comprehensive evidence for a strong inhibition of 5a-reductase activities by mitotane. Importantly,
the strong inhibition of 5a-reductase has significant conse- quences for androgen bioactivity, because the conversion of testosterone to the most potent androgen, 5a-dihydrotestos- terone, will be greatly reduced. Consequently, this may result in enhanced conversion of testosterone to 17ß-estradiol by widespread CYP19A1 (P450 aromatase) activity, which could explain the high incidence of gynecomastia in mito- tane-treated patients. The lack of conversion of testosterone to 5a-dihydrotestosterone also represents a logical explana- tion for the frequent clinical observation of relative ineffi- ciency of testosterone replacement with regard to erectile dysfunction. Our computational analysis of the ratios of 5x- to 5ß-reduced steroids revealed a distinct pattern of global 5a-reductase inhibition by mitotane compared with patients treated with a selective 5a-reductase type 2 inhibitor or pa- tients with inactivating 5x-reductase type 2 mutations. These results could indicate preferential inhibition of 5@-reductase type 1 by mitotane. 5a-Reductase inhibition could also have beneficial consequences in the context of androgen-produc- ing ACC, where it would be likely to help ameliorate the clinical manifestations of androgen excess.
A number of early studies addressed the impact of mi- totane on adrenal steroidogenesis, reporting inhibitory ef- fects of mitotane on 110-hydroxylase, 3ß-hydroxysteroid dehydrogenase, and 18-hydroxylase activities (36-39). However, in vivo studies by labeled isotope infusion were very limited in numbers, whereas in vitro studies were somewhat limited in their methodological approach. In our study, we found no evidence for distinct enzyme in- hibition other than the above described strong inhibition of 5a-reductase activities and the induction of CYP3A4/5. Specifically, there was no change in 11-deoxycortisol me- tabolite excretion, rendering a significant change in 11ß- hydroxylase (CYP11B1) enzymatic activity highly un- likely. However, we observed a down-regulation of overall steroidogenesis as quantified by the sum of total androgen and mineralocorticoid excretion; glucocorticoid metabolites were excluded for that analysis as altered due to the mandatory exogenous hydrocortisone replacement in mitotane-treated patients. These findings could indicate an inhibition of CYP11A1, i.e. P450 side-chain cleavage en- zyme, as previously described in vitro (40), which would result in decreased conversion of cholesterol to pregnenolone and thus a decreased substrate entry flow into the steroido- genic pathways. This could contribute to the hypercholes- terolemia that is a widely documented side effect of mitotane treatment and that has been previously suggested to be due to increased cholesterol synthesis as a consequence of mito- tane-induced up-regulation of 3-hydroxy-3-methyl-glutaryl- coenzyme A-reductase activity (41, 42).
Our study provides a quantifiable measure for the strong induction of CYP3A4/5 activities by mitotane,
A
50
CYP3A4 activity (6BOHF/F)
40
6BOHF/F
30
20
10
0
Reference Range
0
2
4
6
8
10
12
Time (months)
B
5a-reductase activity (5aTHF/THF)
1.4
Reference Range
1.2
5aTHF/THF
1
0.8
0.6
0.4
0.2
0
0
2
4
6
8
10
12
Time (months)
C
5a-reductase activity (An/Et)
1.4
1.2
Reference Range
1
An/Et
0.8
0.6
0.4
0.2
0
0
2
4
6
8
10
12
Time (months)
D
Mitotane Treatment
1.4
6ßOHF/F
40
1.2
30
1
0.8
Plasma
Mitotane (mg/L)
20
0.6
0.4
10
0.2
0
0
0
12
24
36
48
Time (months)
which has the clinically most relevant consequences, in- cluding potential drug interactions in mitotane-treated pa- tients, nicely summarized in a recent review (43). This has an impact not only on drugs needed for the treatment of mitotane-related side effects but, importantly, also on an- titumor drugs including tyrosine kinase and mammalian target of rapamycin inhibitors and also chemothera-
peutic agents included in the current first-line treatment for metastatic ad- renal cancer (44).
Our findings of strong and long-last- ing inhibition of 5a-reductase and induc- tion of CYP3A4/5 refine our understand- ing of the requirements for steroid replacement therapy in mitotane-treated patients. Our observation of a very rapid induction of CYP3A4 by mitotane sug- gests that lower glucocorticoid replace- ment doses, such as 25 mg cortisone acetate (18) equivalent to 15 mg hy- drocortisone (45), may soon become inadequate and contribute to the gas- trointestinal toxicity observed during the first months of mitotane treat- ment. Thus, we suggest that glucocor- ticoid replacement in mitotane- treated patients should be initiated and maintained with at least double the dose normally used in primary ad- renal insufficiency, i.e. 40-50 mg hy- drocortisone (equal to 75 mg cortisone acetate) rather than 20-25 mg hydro- cortisone (equal to 37.5 mg cortisone acetate) per day. Dexamethasone should be avoided because it exerts a strong CYP3A4/5-inducing effect (46) that is likely to result in even more rapid inactivation. Whether urinary 6BOHF excretion can be used as a guide for dose adjustment will have to be examined by prospective studies. At present, the ap- 5&THF/THF propriateness of glucocorticoid re- placement during mitotane therapy largely relies on clinical assessment and plasma ACTH measurements after the morning hydrocortisone dose, with in- creased levels suggestive of glucocorti- coid underreplacement. Importantly, we should consider the use of 5a-re- duced androgens, including synthetic androgens, for androgen replacement in mitotane-induced male hypogonad- ism, which may prove more effective and less prone to unwanted side effects than testosterone replacement therapy. Pregnancy needs to be added to the list of contraindications for mitotane therapy, and patients should have safe contraception in place because the strong inhibition of 5a-reductase activity would have a major impact on sexual differentiation, with a high likelihood of disordered sex development in the male fetus.
Acknowledgments
We are grateful to Amar Agha and Lucy Ann Behan, Beaumont Hospital, Dublin, Ireland, for access to 24-h urine samples from patients receiving regular dose hydrocortisone replacement for adrenal insufficiency.
Address all correspondence and requests for reprints to: Prof Wiebke Arlt MD DSc FRCP FMedSci, Centre for Endocrinology, Diabetes and Metabolism, School of Clinical & Experimental Medicine, University of Birmingham, Birmingham, B15 2TT, United Kingdom, Tel. +44 121 415 8716, Fax +44 121 415 8712, E-mail: w.arlt@bham.ac.uk.
This work was supported by the Medical Research Council UK (Strategic Grant G0801473 to W.A. and P.M.S.), the Euro- pean Union under the Seventh Framework Program (FP7/2007- 2013, Grant Agreement 259735, ENSAT-CANCER), the Na- tional Institute of Health Research UK (NIHR Academic Clinical Fellowship, to V.C.), and the Claire Khan Adrenal Trust Fund.
Disclosure Summary: The authors have nothing to disclose.
References
1. Fassnacht M, Libé R, Kroiss M, Allolio B 2011 Adrenocortical car- cinoma: a clinician’s update. Nat Rev Endocrinol 7:323-335
2. Bergenstal DM, Lipsett M, Moy RH, Hertz R 1959 Regression of adrenal cancer and suppression of adrenal function in man by o,p’- DDD. Trans Am Physicians 72:341-350
3. Hahner S, Fassnacht M 2005 Mitotane for adrenocortical carci- noma treatment. Curr Opin Investig Drugs 6:386-394
4. Hutter Jr AM, Kayhoe DE 1966 Adrenal cortical carcinoma. Results of treatment with o,p’DDD in 138 patients. Am J Med 41:581-592
5. Lubitz JA, Freeman L, Okun R 1973 Mitotane use in inoperable adrenal cortical carcinoma. JAMA 223:1109-1112
6. Jarabak J, Rice K 1981 Metastatic adrenal cortical carcinoma. Pro- longed regression with mitotane therapy. JAMA 246:1706-1707
7. Luton JP, Cerdas S, Billaud L, Thomas G, Guilhaume B, Bertagna X, Laudat MH, Louvel A, Chapuis Y, Blondeau P 1990 Clinical features of adrenocortical carcinoma, prognostic factors, and the effect of mitotane therapy. N Engl J Med 322:1195-1201
8. Wooten MD, King DK 1993 Adrenal cortical carcinoma. Epidemi- ology and treatment with mitotane and a review of the literature. Cancer 72:3145-3155
9. Terzolo M, Angeli A, Fassnacht M, Daffara F, Tauchmanova L, Conton PA, Rossetto R, Buci L, Sperone P, Grossrubatscher E, Re- imondo G, Bollito E, Papotti M, Saeger W, Hahner S, Koschker AC, Arvat E, Ambrosi B, Loli P, Lombardi G, Mannelli M, Bruzzi P, Mantero F, Allolio B, Dogliotti L, Berruti A 2007 Adjuvant mitotane treatment for adrenocortical carcinoma. N Engl J Med 356:2372- 2380
10. Vilar O, Tullner WW 1959 Effects of o,p’DDD on histology and 17-hydroxycorticosteroid output of the dog adrenal cortex. Endo- crinology 65:80-86
11. Kaminsky N, Luse S, Hartroft P 1962 Ultrastructure of adrenal cortex of the dog during treatment with DDD. J Natl Cancer Inst 29:127-159
12. Jensen BL, Caldwell MW, French LG, Briggs DG 1987 Toxicity, ultrastructural effects, and metabolic studies with 1-(o-chloro- phenyl)-1-(p-chlorophenyl)-2,2-dichloroethane(o,p’-DDD) and its methyl analog in the guinea pig and rat. Toxicol Appl Phar- macol 87:1-9
13. Southren AL, Tochimoto S, Strom L, Ratuschni A, Ross H, Gordon G 1966 Remission in Cushing’s syndrome with o,p’-DDD. J Clin Endocrinol Metab 26:268-278
14. Temple Jr TE, Jones Jr DJ, Liddle GW, Dexter RN 1969 Treatment of Cushing’s disease. Correction of hypercortisolism by o,p’DDD without induction of aldosterone deficiency. N Engl J Med 281: 801-805
15. Luton JP, Mahoudeau JA, Bouchard P, Thieblot P, Hautecouverture M, Simon D, Laudat MH, Touitou Y, Bricaire H 1979 Treatment of Cushing’s disease by o,p’DDD. Survey of 62 cases. N Engl J Med 300:459-464
16. Donadille B, Groussin L, Waintrop C, Abbas H, Tenenbaum F, Dugué MA, Coste J, Bertagna X, Bertherat J 2010 Management of Cushing’s syndrome due to ectopic adrenocorticotropin secretion with 1,ortho-1, para’-dichloro-diphenyl-dichloro-ethane: findings in 23 patients from a single center. J Clin Endocrinol Metab 95: 537-544
17. Nader N, Raverot G, Emptoz-Bonneton A, Déchaud H, Bonnay M, Baudin E, Pugeat M 2006 Mitotane has an estrogenic effect on sex hormone-binding globulin and corticosteroid-binding globulin in humans. J Clin Endocrinol Metab 91:2165-2170
18. Daffara F, De Francia S, Reimondo G, Zaggia B, Aroasio E, Por- piglia F, Volante M, Termine A, Di Carlo F, Dogliotti L, Angeli A, Berruti A, Terzolo M 2008 Prospective evaluation of mitotane tox- icity in adrenocortical cancer patients treated adjuvantly. Endocr Relat Cancer 15:1043-1053
19. Robinson BG, Hales IB, Henniker AJ, Ho K, Luttrell BM, Smee IR, Stiel JN 1987 The effect of o,p’-DDD on adrenal steroid replacement therapy requirements. Clin Endocrinol (Oxf) 27:437-444
20. Arlt W, Biehl M, Taylor AE, Hahner S, Libé R, Hughes BA, Sch- neider P, Smith DJ, Stiekema H, Krone N, Porfiri E, Opocher G, Bertherat J, Mantero F, Allolio B, Terzolo M, Nightingale P, Shack- leton CH, Bertagna X, Fassnacht M, Stewart PM 2011 Urine steroid metabolomics as a biomarker tool for detecting malignancy in ad- renal tumors. J Clin Endocrinol Metab 96:3775-3784
21. Hermsen IG, Fassnacht M, Terzolo M, Houterman S, den Hartigh J, Leboulleux S, Daffara F, Berruti A, Chadarevian R, Schlumberger M, Allolio B, Haak HR, Baudin E 2011 Plasma concentrations of o,p’DDD, o,p’DDA, and o,p’DDE as predictors of tumor response to mitotane in adrenocortical carcinoma: results of a retrospective ENS@T multicenter study. J Clin Endocrinol Metab 96:1844-1851
22. Galteau MM, Shamsa F 2003 Urinary 6ß-hydroxycortisol: a vali- dated test for evaluating drug induction or drug inhibition mediated through CYP3A in humans and in animals. Eur J Clin Pharmacol 59:713-733
23. Duda RO, Hart PE, Stork DG 2000 Pattern classification. 2nd ed. New York: Wiley-Interscience
24. Waxman DJ, Attisano C, Guengerich FP, Lapenson DP 1988 Hu- man liver microsomal steroid metabolism: identification of the ma- jor microsomal steroid hormone 6ß-hydroxylase cytochrome P-450 enzyme. Arch Biochem Biophys 263:424-436
25. Ged C, Rouillon JM, Pichard L, Combalbert J, Bressot N, Bories P, Michel H, Beaune P, Maurel P 1989 The increase in urinary excre- tion of 6ß-hydroxycortisol as a marker of human hepatic cyto- chrome P450IIIA induction. Br J Clin Pharmacol 28:373-387
26. Wrighton SA, Brian WR, Sari MA, Iwasaki M, Guengerich FP, Raucy JL, Molowa DT, Vandenbranden M 1990 Studies on the expression and metabolic capabilities of human liver cytochrome P450IIIA5 (HLp3). Mol Pharmacol 38:207-213
27. Kupfer D, Balazs T, Buyske DA 1964 Stimulation by o,p’-DDD of cortisol metabolism in the guinea pig. Life Sci 3:959-964
28. Bledsoe T, Island DP, Ney RL, Liddle GW 1964 An effect of o,p’- DDD on the extra-adrenal metabolism of cortisol in man. J Clin Endocrinol Metab 24:1303-1311
29. Straw JA, Waters IW, Fregly MJ 1965 Effect of o,p’-DDD on hepatic metabolism of phenobarbital in rats. Proc Soc Exp Biol Med 118: 391-394
30. Kupfer D, Peets L 1966 The effect of o,p’DDD on cortisol and hexobarbital metabolism. Biochem Pharmacol 15:573-581
31. Saenger P, Forster E, Kream J 1981 6ß-hydroxycortisol: a nonin-
vasive indicator of enzyme induction. J Clin Endocrinol Metab 52: 381-384
32. van Erp NP, Guchelaar HJ, Ploeger BA, Romijn JA, Hartigh J, Gel- derblom H 2011 Mitotane has a strong and a durable inducing effect on CYP3A4 activity. Eur J Endocrinol 164:621-626
33. Hague RV, May W, Cullen DR 1989 Hepatic microsomal enzyme induction and adrenal crisis due to o,p’DDD therapy for metastatic adrenocortical carcinoma. Clin Endocrinol (Oxf) 31:51-57
34. Hogan TF, Citrin DL, Johnson BM, Nakamura S, Davis TE, Borden EC 1978 o,p’-DDD (mitotane) therapy of adrenal cortical carci- noma: observations on drug dosage, toxicity, and steroid replace- ment. Cancer 42:2177-2181
35. Sparagana M 1987 Primary hypogonadism associated with o,p- ‘DDD (mitotane) therapy. J Toxicol Clin Toxicol 25:463-472
36. Bradlow HL, Fukushima DK, Zumoff B, Hellman L, Gallagher TF 1963 A peripheral action of o,p’-DDD on steroid biotransforma- tion. J Clin Endocrinol Metab 23:918-922
37. Brown RD, Nicholson WE, Chick WT, Strott CA 1973 Effect of o,p’DDD on human adrenal steroid 11ß-hydroxylation activity. J Clin Endocrinol Metab 36:730-733
38. Touitou Y, Bogdan A, Auzeby A, Dommergues JP 1979 Glucocorti- coid and mineralocorticoid pathways in two adrenocortical carcino- mas: comparison of the effects of o,p’-dichlorodiphenyldichloroeth- ane, aminoglutethimide and 2-p-aminophenyl-2-phenylethylamine in vitro. J Endocrinol 82:87-94
39. Ojima M, Saitoh M, Itoh N, Kusano Y, Fukuchi S, Naganuma H 1985 [The effects of o,p’-DDD on adrenal steroidogenesis and he- patic steroid metabolism]. Nihon Naibunpi Gakkai Zasshi 61:168 - 178 (Japanese)
40. Kurokohchi K, Nishioka M, Ichikawa Y 1992 Inhibition mecha- nism of reconstituted cytochrome P-450scc-linked monooxygenase system by antimycotic reagents and other inhibitors. J Steroid Biochem Mol Biol 42:287-292
41. Stacpoole PW, Varnado CE, Island DP 1982 Stimulation of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity by o,p’- DDD. Biochem Pharmacol 31:857-860
42. Maher VM, Trainer PJ, Scoppola A, Anderson JV, Thompson GR, Besser GM 1992 Possible mechanism and treatment of o,p’DDD- induced hypercholesterolaemia. Q J Med 84:671-679
43. Kroiss M, Quinkler M, Lutz WK, Allolio B, Fassnacht M 2011 Drug interactions with mitotane by induction of CYP3A4 metabolism in the clinical management of adrenocortical carcinoma. Clin Endo- crinol (Oxf) 75:585-591
44. Fassnacht M, Terzolo M, Allolio B, Baudin E, Haak H, Berruti A, Welin S, Schade-Brittinger C, Lacroix A, Jarzab B, Sorbye H, Torpy DJ, Stepan V, Schteingart DE, Arlt W, Kroiss M, Leboulleux S, Sperone P, Sundin A, Hermsen I, Hahner S, Willenberg HS, Tabarin A, Quinkler M, de la Fouchardière C, et al. 2012 Combination che- motherapy in advanced adrenocortical carcinoma. N Engl J Med 366:2189-2197
45. Allolio B, Kaulen D, Deuss U, Hipp FX, Winkelmann W 1985 Com- parison between hydrocortisone and cortisone acetate as replace- ment therapy in adrenocortical insufficiency. Aktuelle Endokrinolo- gie Stoffwechsel 6:35-39
46. Villikka K, Kivistö KT, Neuvonen PJ 1998 The effect of dexameth- asone on the pharmacokinetics of triazolam. Pharmacol Toxicol 83:135-138
THE ENDOCRINE SOCIETY®
Members can search for endocrinology conferences, meetings and webinars on the Worldwide Events Calendar. www.endo-society.org/calendar