Ketoconazole Binds to the Human Androgen Receptor

in vivo in vitro in vivo


Introduction
Ketoconazole is a synthetic imidazole with anti-fungal properties. Since it is effective when given orally, it has become widely used for the treatment of a variety of fungal infections, and has become the treatment of choice for both mucocutaneous candidiasis and North American blastomycosis {Dismukes, Stamm, Graybill, Craven, Stevens, Stiller, Sarosi, Medoff, Gregg, Gallis, Fields, Marier, Kerkering, Kaplowitz, Cloud, Bowles and Shadomy 1983;Bradsher, Rice and Abernathy 1985). While therapeutic use of ketoconazole has been infrequently associated with serious side effects (except in patients with previous liver disease), there have been several reports of endocrine dysfunction including, adrenal insufficiency, especially with high dose therapy required in the treatment of life threatening systemic mycoses {Pont, Williams, Azher, Reitz, Bochra, Smith and Stevens 1982a;Pont, Williams, Loose, Feldman, Reitz, Bochra, Smith and Stevens 1982b;Pont, Graybill, Craven, Galgiani, Dismukes, Reitz and Stevens 1984). In men, signs of androgen deficiency, including decreased libido, impotence, gynecomastia, and oligospermia, have been reported in association with the use of ketoconazole. Since decreased testosterone levels have been found in both patients and normal volunteers given ketoconazole, it has generally been assumed that the signs and symptoms of androgen deficiency are secondary to impaired testicular synthesis of testosterone {Grosso, Boyden, Pamenter, Johnson, Stevens and Galgiani 1983;Schurmeyer and Nieschlag 1984;Sikka, Swerdloff and Rajfer 1985;Kan, Hirst and Feldman 1985).
However, it is possible that the drug may be acting via other means of inducing androgen deficiency besides inhibition of testosterone synthesis such as by increasing androgen clearance, as might occur from alterations of testosterone binding to serum binding proteins, or by competition for androgen binding sites on androgen receptors in target tissues. It is possible, for instance, that binding of ketoconazole to sex steroid binding globulin (SSBG) could play a role in the decreased testosterone levels observed in patients taking ketoconazole chronically. In fact, Grosso et al. (1983) have previously shown that ketoconazole displaces steroid hormones from serum transport proteins and, Glass (1986) has shown that one week administration of ketoconazole stimulated a rise in serum LH and FSH levels presumably due to the acute lowering of serum testosterone levels.
Interaction of ketoconazole with those proteins responsible for androgen transport and biological activi-C. Eil ty in vivo could play a role in the pathogenesis of the hypogonadism observed with therapeutic administration of ketoconazole. In this study, the potential interaction of ketoconazole with human androgen receptors was assessed by using an established in vitro assay for measuring the binding of radiolabeled androgens to the androgen binding sites present in cultured human genital skin fibroblasts. The results indicate that ketoconazole possesses affinity for the human androgen receptor protein.

Cell Culture
Fibroblast cultures were established from genital skin specimens obtained from neonates at routine circumcision. The specimens were finely minced in 60 mm petri dishes containing 4-5 ml improved Eagle's Minimum Essential medium (IMEM) supplemented with 10% fetal calf serum, 10-7 M insulin, collagenase (2 mg/ml), penicillin (100 U/ml), streptomycin (100 ug/ml), and glutamine (0.584 g/liter). This medium was freshly prepared for each sample and filtered through a Swinnex 13 Milipore filter (Millipore Corp., Bedford, MA) immediately before use. After mincing, the mixtures were transferred to 25 cm 2 tissue culture flasks and maintained in the presence of 5 % CO2 in a humidified incubator. After 24 h, the medium was changed to a growth medium consisting of IMEM supplemented with the additives listed above except collagenase. When colonies had grown to approximately 50% confluence (usually within 1 -3 weeks), the cells were detached with 0.05% trypsin-0.02% EDTA in PBS at 37 °C and passed serially into larger flasks (75 and 150 cm 2 ).

Whole Cell [ 3 HJR1881 Binding Assay
The binding of imidazoles to the androgen receptor was determined by competitive displacement of [ H]R1881 from receptor sites in cultured human fibroblasts using a dispersed, intact cell assay system as previously described (Eil, Lippman and Loriaux 1980;Eil and Edelson 1984). Briefly, fibroblasts were grown to confluence in five or six 150 cm tissue culture flasks for routine assay. This usually required 4-6 weeks from the time of the initial seeding of the cell line. All studies were performed between passages 3 -20. Two days before assay, the medium was changed to one lacking fetal calf serum. This was repeated again 24 hours before assay. The remainder of the procedure was as previously described (Eil and Edelson 1984), with the binding studies performed at 22 °C to minimize metabolism of the radioligand and competitors, except that [ H]R1881 was substituted for pH]DHT. Competition assays were performed with 0.5 -1.0 nM [ 3 H]R1881 and increasing amounts of the nonradioactive compounds. Binding to low affinity sites was determined in the presence of 5 x 10-7 M R1881 and was subtracted from whole cell binding of [ 3H ]R 1881 obtained in the absence of any inhibitor to assess binding to 5 high affinity sites. All compounds were dissolved in ethanol and added in small aliquots to each tube. The ethanol was allowed to evaporate before addition of the cell suspension and the radioligand, both of which were prepared in IMEM not containing serum or insulin. Although the normal cell line used varied, standard amounts of unlabeled R1881 (0.2, 0.5, 1.0, 2.0 and 500 nM) were included in each assay as an internal control. The amount of R1881 required for 50% displacement of [ 3 H]R1881 was 0.50 ± 0.08 (SE) nM (5 assays), identical to the Kd determined directly with increasing amounts of [ 3 H]R1881 (Eil and Edelson 1984). The percent binding of [ 3 H]R1881 in the absence of unlabelled hormone was 1.0-5.0%; binding to low affinity sites was 5 -10 % of the total bound radioactivity. The number of cells added per tube (0.5-2.0 x 10 ) determined these percentages. Individual data points were determined in duplicate. Binding assays with [ H]dexamethasone were performed in an analogous fashion.

Results
Binding of ketoconazole to the androgen receptor was examined by determining the ability of increasing amounts of the compound to displace [ 3H]R1881 from the androgen receptors in cultured human fibroblasts (Figure 1). While it is clear that ketoconazole can interact with androgen receptors in vitro, a concentration of 6.4+ 1.8 (SE) x 10-M (n = 3) ketoconazole was required for 50% displacement of the tracer from the androgen receptor in this system. Similar results were obtained when the binding assay was performed at 37 °C. The relative binding activity of ketoconazole for binding to the androgen receptor at 22 °C, calculated as the ratio of the concentration of unlabelled R1881 to the concentration of ketoconazole required for 50% displacement of the [ 3 H]R1881 was 7.8 x 10 -6 Scatchard analysis of additional binding studies performed with a fixed amount of ketoconazole (4 x 10 M) and increasing concentrations of [ HJR1881 indicates that ketoconazole interacts with androgen receptors in a competitive fashion (Figure 2). No displacement of [ H]R1881 occurred when other imidazole agents used therapeutically -clotrimazole, miconazole, and fluconazole -were tested in this assay system, at doses up to 4 x 10~4 M each (data not shown).
Ketoconazole also competed for [ H]dexamethasone binding to fibroblast glucocorticoid receptors with a displacement curve parallel to unlabelled dexamethasone (results not shown). Fifty percent inhibition of [ 3 H]dexamethasone binding was achieved by 3 x 10-4 M ketoconazole.

Discussion
With the increasing use of ketoconazole as an oral fungicide, additional aspects of its clinical pharmacology are becoming apparent (Loose, Kan, Hirst, Marcus and Feldman 1983a;Feldman 1986). Due to the fact that ketoconazole is an inhibitor or cytochrome p450 enzyme systems (Sikka, Swerdloff and Rajfer 1985;Sonino 1987), it is not surprising that both adrenal and testicular steroidogenesis, each requiring multiple hydroxylation reactions, are inhibited both in vitro and in vivo. However, the enzyme blockade in vivo must only be partial, since patients on long-term therapy with conventional doses of ketoconazole usually do not develop adrenal insufficiency or hypogonadism, suggesting that the pituitary is able to override the glandular dysfunctions by increasing ACTH and LH secretion. Nonetheless, this inhibitory effect of ketoconazole on steroidogenesis has been exploited clinically to treat successfully both ACTH and non-ACTH dependent hypercortisolism and gonadotropin-independent familial testotoxicosis (Engelhardt, Mann, Hormann, Braun and Karl 1983;Shepard, Hoffert, Evans, Emery and Trachtenberg 1985;Sonino, Boscaro, Merola and Mantero 1985;Contreras, Altieri, Liberman, Gac, Rojas, Ibarra, Ravanal and Seron-Ferre 1985;Holland, Fishman, Bailey and Fazekas 1985).
Based on observations by Loose, Stover and Feldman (1983b) that ketoconazole binds to rat glucocorticoid receptors and Hackney and Sch wartz (1988) that the drug competed for [ H Jdexamethasone binding in a human leukemia cell line, it is also possible that some of ketoconazole's glucocorticoid antagonist activity may be occurring by virtue of occupancy of glucocorticoid receptor sites in target tissues. We confirmed this observation by demonstrating ketoconazole's inhibition of [3H ] dexamethasone binding in dispersed human skin fibroblasts. Because certain drugs which induce gynecomastia, such as cimetidine (Hall 1976) and spironolac-tone (Loriaux, Menard, Taylor, Pita and Santen 1976;Huffman, Kampmann, Hignite and Azarnoff 1978), are believed to have anti-androgenic properties on the basis of their ability to interfere with androgen binding to its receptor (Eil and Edelson 1984), ketoconazole was tested for this property as well. The results of the studies reported here indicate that ketoconazole is a competitive inhibitor of the human fibroblast androgen receptor, albeit with relatively weak affinity. Its potency in this regard is similar to that of cimetidine (Eil and Edelson 1984). While the dose of ketoconazole required for 50 % occupancy of the androgen receptor is not likely to be achieved in vivo in serum by routine anti-fungal therapy, it is possible that the drug may be concentrated in target tissues, such as the breast, allowing for sufficient levels to interact with the receptor. Finally, individuals with diminished metabolic clearance of ketoconazole might achieve particularly high serum levels of the drug while on standard anti-fungal doses. Since there are no progesterone receptors in fibroblasts (Eil, Lippman and Loriaux 1980), it is extremely unlikely that ketoconazole is displacing [ 3 H] R1881 from sites other than androgen receptors. Finally, the data presented here do not distinguish between ketoconazole being an androgen receptor antagonist or agonist. This would require additional studies with bioassays.
It is interesting to note that, while these studies were in progress, Ayub and Levell (1989) reported that ketoconazole along with a number of other imidazole drugs had little or no effect on [ 3 H] R1881 binding to human prostate cytosol androgen receptors. The only possible explanation for the discrepant results between the two studies regarding ketoconazole is that the current assay is performed at 22 °C with intact cells, while the prostate receptor assay is performed at 4 °C with cell extracts. Under our conditions at the higher temperature it is conceivable that ketoconazole is metabolized to a compound with affinity for the androgen receptor. Additional studies at 37 °C confirmed the observations of 22 °C. However, other studies have shown that ketoconazole binds to other receptors and serum binding proteins at 4 °C (Grosso et al. 1983;Loose, Stover and Feldman 1983b;Hodge, Eil, De-Grange, Liberman, Marx and Dunn 1984;Hackney and Schwartz 1988;Ayub and Levell 1989). Furthermore, previous studies with numerous steroids and non-steroidal compounds using the present assay have validated its accuracy and reliability (Eil and Edelson 1984). In any case, from the studies performed here it appears that ketoconazole's interaction with the androgen receptor is competitive and not artifactual.
The demonstration that ketoconazole interacts with glucocorticoid (Loose, Stover and Feldman 1983;Hackney and Schwartz 1988;androgen, and also 1,25-dihydroxyvitamin D (Hodge et al. 1984) receptors, as well as SSBG, suggests that the ligand binding sites of these proteins share some features in common. On the other hand, recent work by Svec (1988) indicates that ketoconazole may interact with the glucocorticoid receptor at a site other than the agonist binding site. The data shown in Figure 2 indicate that, at least for the androgen receptor, ketoconazole appears to act as a competitive inhibitor, most likely at the ligand binding site. In either case, ketoconazole, or other imidazole derivatives, may prove useful in probing the structure and function of steroid and vitamin D receptor proteins, especially now that the primary amino acid sequences of most of these proteins are known.