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Hospital for Children and Adolescents (M.H., L.D., T.R.), University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland; Biomedicum Helsinki (O.A.J., T.R.), Institute of Biomedicine/Physiology, and Department of Clinical Chemistry (O.A.J.), University of Helsinki, Helsinki, Finland; Department of Pediatrics (K.N.-S.), Turku University Central Hospital, Turku, Finland; and Department of Pediatrics (L.D.), Kuopio University Hospital, Kuopio, Finland
Address all correspondence and requests for reprints to: Taneli Raivio, M.D., Hospital for Children and Adolescents, University of Helsinki, P.O. Box 281, FIN-00029 HUS, Finland. E-mail: taneli.raivio{at}helsinki.fi.
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Abstract |
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Objective: The aim of this study was to examine the contribution of flutamide to circulating antiandrogenic activity in children with congenital adrenal hyperplasia using a recombinant cell bioassay.
Design: We describe an open-label, prospective clinical study.
Setting: The study was conducted at the Hospital for Children and Adolescents, University of Helsinki, or the Turku University Hospital, Finland.
Participants: Seven children, age 7.2–10.5 yr, were included.
Intervention: As an experimental approach to improve control of height velocity and the rate of bone maturation, the patients received letrozole (2.5 mg/d) and flutamide (10 mg/kg·d) and were followed up at 3-month intervals for 3–12 months. Before employing the bioassay, two pools of sera (obtained before and during flutamide treatment) were supplemented with increasing amounts of testosterone, and all sera (n = 27) of individual patients were supplemented with a constant amount of exogenous testosterone.
Main Outcome Measure: The main outcome measure was circulating antiandrogenic activity.
Results: Flutamide and/or its metabolites shifted the dose-response curve of testosterone, in that only the highest testosterone concentration, corresponding to 1803 ng/dl (62.5 nM) in human serum, was measurable by the bioassay. In individual sera supplemented with testosterone, flutamide treatment suppressed androgen bioactivity from 378 ± 20 ng/dl (13.1 ± 0.7 nM) (mean ± SEM) (pretreatment) to 110 ± 20 ng/dl (3.8 ± 0.7 nM) (3 months), 83.7 ± 12 ng/dl (2.9 ± 0.4 nM) (6 months), 46.2 ± 6 ng/dl (1.6 ± 0.2 nM) (9 months), and 57.7 ± 9 ng/dl (2.0 ± 0.3 nM) (12 months) testosterone equivalents (P < 0.01).
Conclusions: A dose of flutamide less than 10 mg/kg·d appears sufficient to inhibit AR in children. The recombinant cell bioassay employed herein offers a novel means to monitor the treatment of patients receiving antiandrogens.
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Introduction |
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Flutamide is a prodrug that undergoes first-pass metabolism to produce the biologically active metabolite 2-hydroxyflutamide (14). The extent to which androgen receptor (AR) blockade is achieved with peroral flutamide in pediatric patients is unknown, and varying doses of flutamide have been used in children and adolescents, ranging from 62.5–500 mg/d (15, 16). Flutamide treatment is, however, associated with a minimal, but existing, risk for hepatic failure (17, 18, 19). The mechanism of flutamide-related hepatotoxicity is currently unclear, but it has been hypothesized to be caused by the metabolites of the drug (20, 21). To the best of our knowledge, no investigations exist on this subject in children. Moreover, flutamide decreases cortisol clearance in adult men (22) and children (23), a phenomenon probably reflecting flutamide-induced suppression of hepatic microsomal enzyme activities (22, 24). Although children with CAH might even benefit from prolonged action of hydrocortisone (HC), the dose during flutamide treatment has to be carefully adjusted to minimize the risk for iatrogenic Cushing’s syndrome (23). On the basis of these phenomena, it would seem justified to administer a minimal effective dose of flutamide to children requiring antiandrogen therapy. It is therefore important to know the degree of AR blockade achieved with this drug.
We have recently developed a recombinant cell bioassay that enables measurement of circulating androgen bioactivity directly from a small amount of human serum. This bioassay is based on transient expression of the amino-terminal region and the ligand-binding domain of the AR as separate polypeptides together with AR-interacting protein 3, a steroid receptor coactivator, in COS-1 cells (25). This bioassay detects differences in the bioactivities of naturally occurring androgens and is able to account for the effects of the nonsteroidal antiandrogens bicalutamide and hydroxyflutamide (25). In the current work, we employed this bioassay to investigate the amount of antiandrogenic activity that peroral flutamide administration contributed to circulation of children with CAH.
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Patients and Methods |
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Inclusion criteria for the current investigation were as follows: a salt-wasting or a simple virilizing form of CAH due to 21-hydroxylase deficiency diagnosed on the basis of clinical and biochemical findings; chronological age more than 3 yr; bone age that was more advanced than chronological age; and at least one of the following criteria regarding growth: predicted adult height less than –2 SD; predicted adult height more than 2 SD below mid-parental target height; predicted adult height less than –0.5 SD and weight for height more than +20%. Thus, all patients included to receive this experimental treatment either had severe problems with growth and/or displayed signs of iatrogenic hypercortisolism. Exclusion criteria were bone age more than 14 yr or any disease affecting the liver or kidneys. Eight patients were initially enrolled from the outpatient clinics for pediatric endocrinology at the Hospital for Children and Adolescents, University of Helsinki, or at the Turku University Hospital, Finland. At the start of flutamide and letrozole treatments, all patients received oral HC substitution (8.2–17.6 mg/m2·d), and all except one received oral fludrocortisone (0.1–0.2 mg/d). Of the seven patients included, two received GnRH analog (Procren Depot, Abbott, manufactured in Osaka, Japan) as treatment for central precocious puberty diagnosed previously. Characteristics of the patients at the start of flutamide and letrozole treatment (0 months) are shown in Table 1
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The patients and their guardians were thoroughly informed of the potential benefits and risks of flutamide and the aim of treatment (13). Before the start of treatment, written informed consent was obtained from the patient and his or her guardian. Subsequently, the aromatase inhibitor letrozole (Femar; Novartis AG, Stein, Switzerland) 2.5 mg orally once a day and flutamide (encapsulated by the hospital pharmacy from pulverized Eulexin, Heist-op-den-Berg, Belgium) were started. During the following 2 wk, the flutamide dose was gradually raised from 5 mg/kg·d to the final dose of 10 mg/kg·d divided in two doses; however, a maximum of 500 mg/d was never exceeded. Simultaneously, HC dose was reduced to minimize the risk for iatrogenic hypercortisolism (23). During the rest of the follow-up, HC dose was further reduced based on weight gain, height velocity, and/or serum concentrations of 17-hydroxyprogesterone (17-OHP), androstenedione, or testosterone (Table 2). During follow-up, the patients were clinically examined at 3-month intervals, with special attention to stage of puberty (26) and to clinical signs suggesting androgen action.
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), after 3 months of treatment, exhibited a marginal increase in serum ALAT activity (45 U/liter). After cessation of flutamide and letrozole, serum ALAT normalized in the patient. After cessation of flutamide, HC dose was increased in both subjects, and neither of them displayed rebound effects suggesting androgen action during the posttreatment follow-up. Thus, seven patients (Table 1) without symptoms or laboratory findings necessitating discontinuation of flutamide were followed-up for 3 months and six patients for 12 months. The study protocol was approved by the ethics committees for the Hospital for Children and Adolescents, University of Helsinki, and Turku University Hospital, Finland. The protocol was approved by the National Agency for Medicines.
Immunoassays
All venous blood samples were drawn between 0800 and 1230 h and were allowed to clot; the serum was separated by centrifugation and stored at –20 C or –70 C until analyzed. In patients followed-up in Helsinki (patients 1–5 in Table 1), serum 17-OHP, androstenedione, and testosterone concentrations were measured by RIAs, as previously described (27, 28). In patients followed-up in Turku (patients 6 and 7 in Table 1), serum concentrations of androstenedione and 17-OHP were determined (after extraction of the samples), respectively, by an in-house RIA [measuring range, 0.2–11.5 ng/ml (0.8–40 nM), total coefficient of variation (CV) 
15%] and by a commercially available RIA (Diagnostic Product Corp., Los Angeles, CA) [measuring range, 0.1–12.5 ng/dl (0.4–37.8 nM), total CV 16%]. In these two patients, serum testosterone levels were measured with a commercially available RIA (Spectria Testosterone Coated Tube RIA; Orion Diagnostica, Espoo, Finland), with a detection limit of 14.4 ng/dl (0.5 nM); the interassay CV ranged from 7.0–4.8%.
Androgen bioassay
Sera were available for androgen bioactivity measurements at 3-month intervals from patients 3–5 (during 0–12 months), patient 1 (during 0–9 months), patient 2 (during 0–3 months), and at 6-month intervals from patients 6 and 7 (during 0–12 months). The time of the last flutamide ingestion was available from the guardians for 19 patient visits; the children had taken the drug either in the evening (9–12 h before the serum sample) or in the same morning (2–5 h before the serum sample). Serum androgen bioactivity was measured by the recombinant cell bioassay developed in our laboratory, as previously described (25). To investigate the consequences of flutamide administration on the dose-response curve of testosterone in human serum, testosterone (Sigma-Aldrich, Steinheim, Germany) was added to two pools of sera, each from six subjects, obtained before and after 6 months of flutamide treatment. In short, the sera were thawed, centrifuged through 0.22-µm Spin-X centrifuge filter units (Corning Costar Corp., New York, NY), and pooled. The testosterone was first dissolved in ethanol, then serially diluted in glass tubes, and 2 µl of each dilution was added to 18 ml of phenol red-free DMEM. These media were subsequently used to dilute aliquots of the two serum pools 1:10. The final added testosterone concentrations in the diluted sera, and, thus, in the culture media of the bioassay, were 2.9 ng/dl (0.1 nM), 11.3 ng /dl (0.39 nM), 22.5 ng/dl (0.78 nM), 45.0 ng/dl (1.56 nM), 89.4 ng/dl (3.1 nM), and 180 ng/dl (6.25 nM). The diluted samples were vortexed and incubated at room temperature for 30 min and at 37 C for 30 min; 100 µl of each diluted sample in triplicate was added to the bioassay wells containing COS-1 cells transiently transfected with the requisite plasmids of the bioassay (25). After an overnight incubation at 37 C, the cells were lysed, and ß-galactosidase (indicator for transfection efficiency) and luciferase (reporter gene) activities in the lysates were measured as previously described (25). The antiandrogenic activity contributed by peroral flutamide was further investigated by supplementing all sera (n = 27) with a constant amount of exogenous testosterone; testosterone was added (in 2 µl of ethanol) to 18 ml of phenol red-free DMEM to achieve a concentration of 200 ng/dl (6.94 nM). Subsequently, 225 µl of the testosterone-containing DMEM was added to 25 µl of each sterile filtered serum sample to achieve a final testosterone concentration of 180 ng/dl (6.25 nM). Then 100 µl of each diluted serum sample in duplicate was subjected to the bioassay. The results were calculated against a standard curve of testosterone in charcoal-stripped fetal calf serum (FCS), and the results are expressed in nanograms per deciliter (nanomolar) testosterone equivalents (25).
Statistics
Paired t test or Wilcoxon rank sum test was employed when appropriate. Because the same subjects were investigated repeatedly, we employed the method of summary measures (29). Arithmetic mean of bioactivities in testosterone-supplemented samples, obtained 2–5 or 9–12 h after flutamide ingestion, was calculated for each subject. In four patients, both average values were available and used as raw data for a paired t test. Values are expressed as mean ± SEM. P < 0.05 was chosen to indicate statistical significance.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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). Patients 2 and 4 (Table 1) displayed the highest 17-OHP levels and Tanner stage II. In addition, patient 4 presented with adult-type sweat odor and comedones, suggesting significant androgen action in the skin. Overall, initiation of flutamide and letrozole treatments brought about a significant decline in serum 17-OHP and androstenedione levels between 0 and 3 months of treatment, despite the concomitant reduction in HC dose (Table 2). Serum 17-OHP levels, measured at 0 and 12 months, did not differ (Table 2). After 3 months of treatment, the clinical signs of androgen action in patient 4 had resolved, except for pubic hair, which disappeared after 12 months of flutamide and letrozole treatments.
All patients displayed undetectably low serum androgen bioactivity [<23.1 ng/dl (0.8 nM) testosterone equivalents] before and during the treatment with flutamide and letrozole. We next evaluated the degree of circulating antiandrogenic activity achieved with peroral flutamide by investigating two pools of sera, each from six subjects, obtained before and after 6 months of flutamide therapy, both of which were supplemented with increasing concentrations of testosterone. Ingested flutamide and/or its metabolites contributed antiandrogenic activity to the serum, because the dose-response curve of testosterone in this serum pool was shifted to the right (Fig. 1) and only the highest testosterone concentration tested [180 ng/dl (6.25 nM) in culture medium] activated the reporter gene in the bioassay (Fig. 1). The dose-response curves of testosterone in FCS and in human serum were parallel (Fig. 1).
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). The highest androgen bioactivity levels (mean ± SEM) [378 ± 20 ng/dl (13.1 ± 0.7 nM) testosterone equivalents] were found in the testosterone-supplemented samples obtained before the start of flutamide therapy. However, the mean androgen bioactivity levels, measured during flutamide treatment (at 3, 6, 9, and 12 months), were all lower than the mean of pretreatment values (P < 0.01) (Fig. 2). We next evaluated the relationship between the time of ingestion of flutamide and circulating antiandrogenic activity in these testosterone-supplemented samples. Compared with pretreatment values, the samples obtained 9–12 h after the preceding flutamide dose tended to display diminished androgen bioactivity [89.4 ± 14 ng/dl (3.1 ± 0.5 nM) testosterone equivalents], and this suppression appeared even stronger [60.6 ± 14 ng/dl (2.1 ± 0.5 nM) testosterone equivalents; P = 0.06; n = 4] in samples drawn shortly (2–5 h) after the ingestion of the drug.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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We estimated circulating antiandrogenic activity during peroral flutamide treatment by adding increasing amounts of testosterone to patient sera obtained before or during flutamide treatment. Surprisingly, flutamide and/or its metabolites, present in serum after the antiandrogen administration, permitted only the highest added testosterone concentration, corresponding to 1803 ng/dl (62.5 nM) in human serum, to activate the reporter gene of the bioassay. Such high endogenous testosterone levels were not encountered in our patients. In contrast, during the course of the treatment, circulating androstenedione, 17-OHP, and testosterone levels did not increase significantly compared with pretreatment values, despite the reduction in HC dose. This finding suggests a significant flutamide-induced decrease in cortisol clearance (23) and a subsequent suppression of adrenal biosynthesis of androgens and their precursors. In adults, the peak in serum hydroxyflutamide concentration occurs 2.6 h after flutamide administration (31). In agreement with this finding was that circulating antiandrogenic activity in our patients after 2–5 h of flutamide ingestion tended to be even stronger than was antiandrogenic activity 9–12 h after ingestion of the drug. On the basis of our results, we suggest that a dose of flutamide less than 10 mg/kg·d is sufficient to inhibit AR function in children with CAH. Indeed, in women with hirsutism, a smaller relative dose of flutamide (250 mg/d) than what we employed in the current investigation has been shown to suppress AR activity (32), and an even smaller relative dose has been clinically effective (33).
It should be noted that all patients displayed undetectably low serum androgen bioactivity levels even before the start of flutamide treatment, although some had unequivocally elevated serum 17-OHP and androstenedione concentrations. This bioassay result was not unexpected, because androstenedione displays only weak activity in the bioassay (25) and has clearly higher dissociation constant (Kd) for the ligand-binding domain of AR [18,690 ng/dl (648 nM)] (34) than its circulating concentrations in the participants of the current work. This suggests that circulating levels of 17-OHP or androstenedione do not significantly activate interaction between the two termini of AR and do not directly account for the clinical signs of androgen action. Instead, it is probable that these compounds serve as substrates for the formation of more potent androgens in several peripheral, nonendocrine tissues (34, 35). This phenomenon potentially explains the simultaneous presence of low circulating androgen bioactivity and clinical signs of androgen action in one of our patients before the start of flutamide therapy. On the other hand, the rapid disappearance of comedones in this female patient during the first 3 months of treatment probably reflected flutamide-induced inhibition of AR function, enhanced suppression of adrenal androgen biosynthesis due to a flutamide-induced decrease in cortisol clearance, or both.
To the best of our knowledge, only one study with a relatively small number of patients with CAH has explored the effects of simultaneous flutamide and aromatase inhibitor (testolactone) treatment in children; 2-yr treatment with these drugs was not associated with severe adverse effects (12). Given the potential hepatotoxicity of flutamide, however, and the lack of long-term safety data, it should be underscored that the treatment regimen employed in the current work remains experimental and, in our opinion, should not be used outside a research setting.
In conclusion, our results in seven children with CAH show that treatment with flutamide provides strong antiandrogenic activity in the circulation and suggest that a dose of flutamide less than 10 mg/kg·d is sufficient to achieve significant inhibition of AR function. The recombinant cell bioassay employed in this work offers a novel means to monitor treatment of patients receiving antiandrogenic therapy.
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Footnotes |
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First Published Online June 28, 2005
1 L.D. and T.R. contributed equally to this study. 
Abbreviations: ALAT, Alanine aminotransferase; AR, androgen receptor; CAH, congenital adrenal hyperplasia; CV, coefficient of variation; FCS, fetal calf serum; HC, hydrocortisone; 17-OHP, 17-hydroxyprogesterone.
Received February 14, 2005.
Accepted June 20, 2005.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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, , -trifluoro-2-methyl-4'-nitro-m-propionotoluidide (Flutamide), in men following a single oral 200 mg dose. J Clin Endocrinol Metab 41:373–379This article has been cited by other articles:
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  P. Roy, M. Alevizaki, and I. Huhtaniemi In vitro bioassays for androgens and their diagnostic applications Hum. Reprod. Update, January 1, 2008; 14(1): 73 - 82. [Abstract] [Full Text] [PDF]
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