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Original Studies |
Department of Pediatrics, Division of Endocrinology, IWK Grace Health Centre (E.A.C., S.R.S.); Department of Pathology (M.L.G.); Department of Medicine, Division of Endocrinology (R.S.R.). Dalhousie University, Halifax, Nova Scotia, Canada B3J 3G9
Address all correspondence and requests for reprints to: Sonia R. Salisbury, M.D., Department of Pediatrics, IWK Grace Health Centre, 5850 University Avenue, Halifax, Nova Scotia, Canada, B3J 3G9.
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Abstract |
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Introduction |
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-oxa-D-homo-androsta-1, 4-diene- 3, 17-dione),
an aromatase inhibitor that blocks conversion of
androgens to estrogens, has been used to treat gonadotropin independent
precocious puberty in girls with and without features of
McCune-Albright syndrome (polyostotic fibrous dysplasia) (1, 2, 3, 4) and
boys with familial male precocious puberty (testotoxicosis) (5, 6). In
boys, testolactone and spironolactone have been used in combination;
however, most of the beneficial effects on growth velocity and bone
maturation were seen with testolactone alone, compared with
spironolactone alone (5). We report 2 cases of gonadotropin independent precocious puberty treated with testolactone. During the course of testolactone treatment, a 2-yr-old girl was found to have markedly elevated levels of androstenedione and testosterone but had no evidence of virilization. Subsequently, a 3-yr-old boy treated with testolactone for familial male precocious puberty also had markedly elevated levels of androstenedione and persistently high testosterone. This study was undertaken to determine whether the elevation in androgens was caused by a true pharmacologic aromatase inhibitor effect of testolactone or was caused by interference by testolactone in the androgen assays. Steroid levels were measured after a single oral dose of testolactone in the two patients and a postmenopausal control. Solutions of testolactone were also added to the androgen assays to determine possible interference in the androstenedione and testosterone serum RIAs.
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Materials and Methods |
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A 1-yr 11-month-old child, with no café-au-lait spots or bony deformity, presented with the sudden onset of bilateral breast development and white vaginal discharge. Her height was on the 50th percentile, her weight was between the 25th and 50th percentiles, and her bone age was normal. Pelvic ultrasound showed a seemingly mature uterus and cysts in the enlarged right ovary. Hormonal investigations included a normal baseline LH at 0.7 IU/L (normal < 1.0) with a flat GnRH stimulation test (peak 0.4 IU/L, after 2 µg/kg GnRH) and a markedly elevated 17ß-estradiol level of 108 pg/mL (normal < 20). A diagnosis of non-McCune Albright gonadotropin independent precocious puberty was made. Androstenedione and testosterone were not measured, but dehydroepiandrosterone sulfate (DHEAS) was normal at 77 ng/mL (normal: 52–722). Over the next 8 months, breast development continued, growth velocity increased to 20 cm/yr (normal: 9 cm/yr), and menses occurred for 13/24 weeks, despite medroxyprogesterone acetate therapy, up to 10 mg tid. Testolactone (20 mg/kg/day, in 4 divided doses) was started, and menses stopped within 1 week. After 3.5 months of testolactone therapy, there was marked regression of pubertal signs and no further vaginal bleeding, and estradiol levels were undetectable. To assess the impact of testolactone as an aromatase inhibitor, androstenedione was measured and discovered to be markedly elevated at 18 ng/mL (normal: 0.2–3.1). Testosterone on the same sample was 3.6 ng/mL (normal < 0.2), but the child had no evidence of virilization. These values were confirmed on repeat testing, 6 weeks later, and the dihydrotestosterone level was also elevated at 3.2 ng/mL (normal < 0.15).
Case 2
This otherwise healthy boy presented at 3.2 yr of age with a history of gradual onset of pubertal changes, including a deep voice, a mature phallus with 5-mL testes (normal < 4 mL) bilaterally, and Tanner stage III pubic hair. His height was above the 97th percentile (giving a height age of 5 yr), and his bone age was advanced (at 7.5 yr). Family history was remarkable for precocious puberty in 7 males in 4 generations of the extended family. Baseline investigations showed a high serum testosterone of 2.3 ng/mL (normal < 0.2), a normal prepubertal DHEAS (at 144 ng/mL) and a low baseline LH of 0.4 IU/L, with an insignificant stimulation to 1.5 IU/L after GnRH (2 µg/kg); and these were consistent with gonadotropin independent precocious puberty. The patient was treated with testolactone [15 mg/kg/day, and gradually increased to 40 mg/kg·day in combination with spironolactone (2 mg/kg/day]. Spironolactone was stopped after 3 weeks, because of gastrointestinal side effects. On testolactone alone, serum testosterone remained elevated (1.9 ng/mL). Because of noncompliance, pubertal signs continued to progress.
In vivo studies
In the course of investigation of the elevated androgen levels in these patients, the following studies were performed. The effect of a single dose of testolactone on testosterone and androstenedione levels was evaluated in the two patients and in a healthy postmenopausal control subject (estradiol < 20 pg/mL; testosterone < 0.2 ng/mL; androstenedione, 1.3 ng/mL). Informed consent was obtained from the parents and the control subject. Testolactone, provided by Bristol Myers Squibb (Teslac, St. Laurent, QC, Canada), was withheld from cases 1 and 2 for at least 12 h preceding the test. Baseline androstenedione and testosterone were drawn at 0800 h; and the single, usual oral dose of testolactone was given (case 1: 5 mg/kg; case 2: 4 mg/kg; control: 10 mg/kg). With an indwelling iv catheter in place, androstenedione was measured hourly for 12 h and at 24 h, and testosterone was measured every 1–3 h for 12 h and at 24 h. Serum estradiol also was measured hourly in the control subject.
Laboratory studies
Serum concentrations of steroids were determined using kits as follows: androstenedione, Sanofi DSL Coated-Tube RIA, Diagnostic Systems Laboratories Inc. (Webster, TX); testosterone, Medicorp ImmuChem Double Antibody RIA, ICN Biomedicals, Inc. (Costa Mesa, Ca); estradiol, Intermedico DPC Coat-a-Count RIA (Intermedico/DPC, Los Angeles, CA); LH, Chiron Diagnostics Chemiluminescence Immunoassay (Chiron, Walpole, MA); DHEAS, Intermedico DPC Coat-a-count DHEA-SO4 RIA (Intermedico/DPC); and dihydrotestosterone, DSL Coated-Tube RIA, Diagnostic Systems Laboratories, Inc. The lower limit of detection for androstenedione was 0.03 ng/mL, with inter- and intraassay coefficients of variation of 6.0% and 4.3%, respectively; and for the testosterone assay, the detection limit was 0.1 ng/mL, with inter- and intraassay coefficients of variation of 9.6% and 9.3%, respectively. The sensitivity of the estradiol assay was 20 pg/mL.
To determine the effect of testolactone on the androgen assays, a 50-mg tablet of testolactone was dissolved in 50 mL of 95% ethanol. Ten and 100 µL of this testolactone solution were added to 50 µL of normal serum to give final concentrations of 0.66 and 6.66 mmol, respectively. These are equivalent to 200 µg/mL and 2000 µg/mL, respectively. The control serum and the two spiked serum solutions were assayed for testosterone and androstenedione.
To further define the cross-reactivity between testolactone and testosterone in the testosterone assay, 50 mg testolactone (one tablet) were dissolved in ethanol, and serial dilutions were made to give final dilutions in the testosterone assay of 0.1–10 ng/mL. The samples were then processed identically to the testosterone standard curve.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
Testosterone levels reached a mean peak of 6.9 ng/mL and returned
to baseline within 24 h (Fig. 1B). Estradiol levels remained
unmeasurable at all sampling points during the 24-h period, in the
control subject.
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demonstrates the nonparallel
testosterone and testolactone curves in the testosterone assay,
confirming cross-reactivity of testolactone in this system. For
example, at a testolactone concentration of 445 ng/mL, a false value of
6.9 ng/mL of testosterone would be measured.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There are two previous studies reporting large elevations in androgens in subjects treated with testolactone. The first reported a 62% increase in 17-ketosteroid excretion, but no virilization, in postmenopausal women treated with 150 mg/day testolactone for metastastic breast cancer (10). In adolescent gynecomastia, treatment with testolactone (450 mg/day) was associated with markedly elevated levels of androstenedione (9.5–21 ng/mL) (11). Testosterone levels in these patients also increased significantly, by approximately 1.5 times the baseline, during testolactone therapy but returned to baseline when testolactone was discontinued. In all of these studies, the androgens were measured by RIA. Interference by testolactone in the assays for testosterone and androstenedione could possibly explain all of these findings.
Other reports of testolactone use in various patient groups were not associated with elevations in androgen levels, aside from small increases that could be consistent with the mechanism of action of testolactone. Significant increases in testosterone and modest, but significant, increases in androstenedione were noted in oligospermic men treated with testolactone (12). However, only slight increases in testosterone, dihydrotestosterone, and androstenedione were measured in eight women with polycystic ovarian disease, given testolactone (500 mg every 6 h). In this study, serum was pooled from samples drawn at 10-min intervals throughout the night and was processed by column chromatography before performance of the RIA (13). These differences in sampling and assay techniques may explain the minimal elevation of androgens found in this study. Judd et al. (14) found only small increases in testosterone, androstenedione, and estradiol (measured by RIA) in postmenopausal women taking testolactone for metastatic breast cancer; however, it is unclear when the blood samples were drawn, in relation to the testolactone dose. They concluded that the changes in testosterone and androstenedione were caused by the mechanism of action of testolactone, whereas the estradiol increase was caused by testolactone cross-reactivity in the estradiol assay. No studies of cross-reactivity of testolactone in the testosterone and androstenedione assays were reported (14). In the present study, there was no increase in estradiol associated with testolactone. Two studies of testolactone use in boys with gonadotropin independent precocious puberty did not report further elevations in testosterone above baseline (5, 6). A recent report of testolactone use in patients with congenital adrenal hyperplasia did not report elevated androgen levels in those patients taking testolactone; however, blood samples were drawn before administration of the morning medications (15).
Testosterone and androstenedione levels, measured by RIA, in patients on testolactone therapy do not represent the true serum values; however, androgens measured in other assay systems may not be subject to the same cross-reactivity as found in these RIAs. The choice of antibody will dictate the degree of cross-reactivity. When testolactone was stopped for 24 h before testing, androgen levels in these patients returned to baseline. In girls, serum estradiol levels seem to reflect endogenous levels, as there is no assay interference. In conclusion, because of interference in these androgen RIAs, response to testolactone in boys must be judged on clinical grounds, and elevations of androgens, in the absence of virilization in girls, must be ignored.
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Footnotes |
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2 Recipient of an Eli Lilly Canada Pediatric Endocrinology
Fellowship.
Received July 23, 1997.
Revised November 24, 1997.
Accepted December 1, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-testolactone(teslac)
in vivo in rats and in vitro in human cultured
fibroblasts, rat mammary carcinoma cells and rat prostate cytosol. Endocrinology. 110:214–219.[Abstract]
1-testololactone on clinical course and
hormonal excretion. Cancer. 15:633–635.[CrossRef]
1-testolactone on the pituitary-testicular axis in
oligospermic men. J Clin Endocrinol Metab. 52:897–902.[Abstract]
1-testolactone on
gonadotropin release and steroid metabolism in polycystic ovarian
disease. J Clin Endocrinol Metab. 60:773–780.[Abstract]
1-testololactone on peripheral aromatization. Cancer
Res. [Suppl]42:3345–3348.
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