Clinical Endocrinology (1991) 35, 163-168
ADONIS 0300066491001 15s
The influence of ketoconazole on human adrenal steroidogenesis: incubation studies with tissue slices D. Engeihardt, M. M. Weber, 1.Miksch, F. Abedinpour and C. Jaspers Medical Department 11, Klinikum GroBhadern, University of Munich, FR Germany (Received 17 December 1990; returned for revision 14 January 1997; finally revised 11 March 1991; accepted 26 March 1997)
Summary OBJECTIVE The influence of ketoconazoie on the varlous enzymes of human adrenal steroid biosynthesls was examined in vitro. MEASUREMENTSAfter incubation of human adrenal tissue slices wlth labelled precursors and ketoconazole(0-2000 pm), radloactive metabolites were separated by thin-layer chromatography and quantified by liquid sclntillation counting. Enzyme activity was assessed by measuring conversion of trltium-labelled precursors to products. RESULTS In virro, ketoconazoie showed a slgnlficant inhibition on the following adrenal enzyme systems (wlth decreasing activity): C17,2O-desmolase (IC= 2 pm), 16a-hydroxylase (I& 9 p ~ )l7a-hydroxylase , (lCso18 pm), 18-hydroxyiase (iCw 28 pm), and llp-hydroxylase (ICw 35 p r ) . In the tested concentrations ketoconazoie had no inhibitory effect on the 21-hydroxylase , the 3/.?-hydroxysteroid dehydrogenase and the 20-hydroxysteroiddehydrogenase component of the C17,ZO-desmolase enzyme system. CONCLUSIONS The data are in accordance wlth clinical findings where a strong suppresslon of serum androgen levels by relatively selective inhibition of C17, 20-desmolase has been assumed. The predominant blocking effect of ketoconazole on adrenal as well as on gonadal androgen biosynthesis might be of clinical benefit In the management of hyperandrogenic states.
The imidazole derivative ketoconazole, an orally active broad-spectrum antimycotic drug, inhibits at picomolar concentrations C 14-methylation of lanosterol to ergosterol and thus disturbs fungal membrane growth (Vanden Bossche et al., 1990). At higher concentrations, the drug also affects Correspondence: Professor Dr med. D. Engelhardt, Medizinische Klinik 11, Klinikum GroDhadern, Universitat Miinchen, MarchioninistraBe 15, 0-8000 Miinchen 70, FR Germany.
steroid biosynthesis in man. The endocrine potencies of the substance result from an interaction of the imidazole ring with the cytochrome P-450 component of various mammalian steroidogenic enzyme systems (Fig. 1). In vivo, the administration of low doses of ketoconazole leads to a significant reduction in serum androgen levels (Santen et al., 1983) while at higher doses castration levels are achieved and cortisol secretion is suppressed (Trachtenberg et al., 1983). This inhibitory effect of ketoconazole on steroid biosynthesis has led to its therapeutic use in the treatment of advanced prostatic cancer (Trachtenberg et al., 1983), hirsutism (Martikainen et al., 1988), precocious puberty (Holland et al., 1985), and Cushing's syndrome (Sonino et al., 1985; Loli et al., 1986; McCance et al., 1987; Engelhardt et al., 1989). From clinical data it has been supposed that the C17,20desmolase, the key enzyme of androgen biosynthesis, is most sensitive to the blocking effect of ketoconazole (Santen et al., 1983). To a lesser extent an inhibition of the 17a-hydroxylase, the 1 Ij-hydroxylase and the 18-hydroxylase has been postulated (De Coster et a[., 1987). While the site of action of ketoconazole with a relative selective inhibition of C17,20desmolase has been confirmed in rat testicular tissue (Lambert et al., 1986; Ayub & Levell, 1987; Vanden Bossche et al., 1990), the results of in-vitro studies with human adrenals remain conflicting. The aim of this study therefore was to investigate the invitro effect of ketoconazole on seven important steroidogenic enzyme systems in human adrenal tissue. Materials and methods Materials
The radiolabelled steroids 1,2,6,7-3H-progesterone (1 12 Ci/ mmol), 1,23H- 17a-hydroxyprogesterone (50 Ci/mmol), 1,23H-17a-hydroxypregnenolone (12 Ci/mmol), I , P H - I 1deoxycortisol(58.6 Ci/mmol), 4-'4C-corticosterone (50 mCi/ mmol), 4-14C-cortisol(55.5 mCi/mmol), 4-14C-1 1-deoxycorticosterone (58.5 mCi/mmol), 4J4C-1 1-deoxyeortisol(50-60 mCi/mmol), 4-14C-progesterone (50-60 mCi/mmol), 4-14C17a-hydroxyprogesterone (50-60 mCi/mmol) and 4J4Candrostenedione (52 mCi/mmol) were purchased from New England Nuclear, Boston, USA. 1,2-3H-corticosterone (49.7 Ci/mmol) was from Amersham Buchler, Braunschweig, FRG, all non-radioactive steroids were obtained from Sigma, Tauflcirchen, FRG. Thin-layer chromatography (TLC) plates Kieselgel 60 (F254) were obtained from Carl 163
164
D.Engelhardt et al.
319 HSD
Clinical Endocrinology (1991) 35
30 HSD 20a HSD
17 OH
110H CORTEOSTERONE
17,20 D
1
30 HSD
110H CORTISOL
18OH 18-OH-CORTICOSTERONE
1
A LD0STER0NE
Flg. 1 Main pathways of adrenal steroid biosynthesis. SCC denotes side-chain cleavage, HSD hydroxysteroid dehydrogenase, OH hydroxylase and D desmolase. Black bars indicate inhibition by ketoconazole.
Roth KG, Karlsruhe, FRG, ketoconazole (Nizoral) was purchased from Janssen GmbH Beerse, Belgium. Preparation and incubation of adrenal tissue
Human adrenal glands were obtained from 18 patients, who underwent adrenalectomy due to renal carcinoma (n= 13), renal transplantation (n= l), phaeochromocytoma (n= 3) or Cushing’s syndrome (n= 1). Adrenal tissue used for incubation experiments was macroscopically and histologically normal except in the patient with Cushing’s syndrome, who had adrenal hyperplasia. Immediately after aseptic removal, the adrenal glands were placed in ice-cold 0.9% NaCl solution and transferred to the laboratory. Slices of 100 mg wet weight were prepared at 4°C after the method of Deutsch (1936) and transferred to incubation vessels containing 2 ml of standard incubation mixture (Engelhardt et al., 1985). The mean & SEM protein content of 100 mg tissue slices, measured by the method of Lowry et al. (19Sl), was 10.9 k0.5 mg (n=9). After addition of radiolabelled precursors and various concentrations of ketoconazole (0-2000 p ~ ) the , adrenal tissue was incubated for 2 hours at 37°C in a shaking waterbath. The reaction was stopped by freezing at -40°C. All incubation experiments were done in triplicate. In preliminary experiments we found a two to three-fold stimulation of steroid biosynthesis by adrenal tissue after the addition of ACTH. Furthermore, the amounts of steroids
formed were related linearly to the amount of adrenal tissue, tissue protein and incubation time.
Analysis of steroid metabolism
The extraction and identification of tritium labelled metabolites were performed as described previously (Engelhardt et al., 1985). Before determination of steroids the tissue was homogenized mechanically and traces of 14C-labelledsteroids (10’ c.p.m.) were added for recovery calculations. After extraction with chloroform and distribution between methanol/n-heptane (2: 1 v/v), the labelled steroids and the metabolites were separated by thin-layer chromatography using the following systems, modified from the method of Neher (1964): chloroform/methanol (95 :5 v/v) and butylacetate/ ethylacetate/isoamylalcohol (1 : 1 : 1 by vol.) for incubation studies with )H-progesterone, )H-l 7a-hydroxyprogesterone and 3H-17a-hydroxypregnenolone;acetone/benzene (1 : 1 v/ v) and n-heptane/benzene/ethylacetate(1 : 1 :4 by vol.) for incubations with 11-deoxycortisol and corticosterone. If necessary, rechromatography was performed with the systems benzene/acetone (1 : 1 v/v) for separation of 17ahydroxyprogesterone, chloroform/methanol (95 :5 v/v) for separation of 16a-hydroxyprogesterone and I 1-deoxycorticosterone, chloroform/ethylacetate (8 :2 v/v) for separation of 17a,20a-dihydroxyprogesterone,androstenedione and
Ketoconazole and human adrenals
Clinical Endocrinology (1991) 35
progesterone. The labelled steroids on the thin-layer plates were localized by added non-labelled steroids. Quantitative analysis was made by liquid scintillation counting. C17,20desmolase activity was estimated by measuring the conversion of the added precursor 3H-17u-hydroxprogesterone (I 317 x mol) to 3H-17a,20a-dihydroxyprogesteroneand 3H-androstenedione. The 17a-hydroxylase activity was assessed by measuring the conversion of the added precursor mol) to 'H-17a-hydroxypro'H-progesterone (4-14 x gesterone, 16a-hydroxylase and 21-hydroxylase activity by measuring the amount of 3H-16u-hydroxyprogesterone and 'H-I 1-deoxycorticosterone in the same incubation experiments. Furthermore, the effect of ketoconazole on the followingenzyme activities was investigated: on 1ljl-hydroxylase by measuring the conversion of the precursor 3H-1I mol) to 'H-cortisol, on 18deoxycortisol (19-34x hydroxylase by measuring the transformation of added 3Hcorticosterone (20-22 x mol) to 'H-18-hydroxycorticosterone and on 21-hydroxylase by measuring the conversion of 3H-17a-hydroxyprogesterone (35-45 x mol) to 3H-lI-deoxycortisol.
165
I20
600
100
500
80
400
," 60
300
5-
+
u
0
p 4-
c
0 0
8 Ol
ES
40
200
20
I00
0,
?
2
I
I
I
1
0
Flg. 2 Effect of increasing concentrations of ketoconazole on A,
the amount of unmetabolized 3H-17~-hydroxyprogesterone; 0, formation of H- 17a,20a-dihydroxyprogesterone; and 0 , 'Handrostenedione after incubation of human adrenal tissue with 3H-17a-hydroxyprogesterone.100% control activity represents amounts of 83.8*8, 17.3+3 or 56.9&4 fmol per mg wet-weight and hour of incubation without ketoconazole. Values are means f SEM of three independent incubation experiments. NS, not significant;* PcO.05; ** P
ADONIS 0300066491001 15s
The influence of ketoconazole on human adrenal steroidogenesis: incubation studies with tissue slices D. Engeihardt, M. M. Weber, 1.Miksch, F. Abedinpour and C. Jaspers Medical Department 11, Klinikum GroBhadern, University of Munich, FR Germany (Received 17 December 1990; returned for revision 14 January 1997; finally revised 11 March 1991; accepted 26 March 1997)
Summary OBJECTIVE The influence of ketoconazoie on the varlous enzymes of human adrenal steroid biosynthesls was examined in vitro. MEASUREMENTSAfter incubation of human adrenal tissue slices wlth labelled precursors and ketoconazole(0-2000 pm), radloactive metabolites were separated by thin-layer chromatography and quantified by liquid sclntillation counting. Enzyme activity was assessed by measuring conversion of trltium-labelled precursors to products. RESULTS In virro, ketoconazoie showed a slgnlficant inhibition on the following adrenal enzyme systems (wlth decreasing activity): C17,2O-desmolase (IC= 2 pm), 16a-hydroxylase (I& 9 p ~ )l7a-hydroxylase , (lCso18 pm), 18-hydroxyiase (iCw 28 pm), and llp-hydroxylase (ICw 35 p r ) . In the tested concentrations ketoconazoie had no inhibitory effect on the 21-hydroxylase , the 3/.?-hydroxysteroid dehydrogenase and the 20-hydroxysteroiddehydrogenase component of the C17,ZO-desmolase enzyme system. CONCLUSIONS The data are in accordance wlth clinical findings where a strong suppresslon of serum androgen levels by relatively selective inhibition of C17, 20-desmolase has been assumed. The predominant blocking effect of ketoconazole on adrenal as well as on gonadal androgen biosynthesis might be of clinical benefit In the management of hyperandrogenic states.
The imidazole derivative ketoconazole, an orally active broad-spectrum antimycotic drug, inhibits at picomolar concentrations C 14-methylation of lanosterol to ergosterol and thus disturbs fungal membrane growth (Vanden Bossche et al., 1990). At higher concentrations, the drug also affects Correspondence: Professor Dr med. D. Engelhardt, Medizinische Klinik 11, Klinikum GroDhadern, Universitat Miinchen, MarchioninistraBe 15, 0-8000 Miinchen 70, FR Germany.
steroid biosynthesis in man. The endocrine potencies of the substance result from an interaction of the imidazole ring with the cytochrome P-450 component of various mammalian steroidogenic enzyme systems (Fig. 1). In vivo, the administration of low doses of ketoconazole leads to a significant reduction in serum androgen levels (Santen et al., 1983) while at higher doses castration levels are achieved and cortisol secretion is suppressed (Trachtenberg et al., 1983). This inhibitory effect of ketoconazole on steroid biosynthesis has led to its therapeutic use in the treatment of advanced prostatic cancer (Trachtenberg et al., 1983), hirsutism (Martikainen et al., 1988), precocious puberty (Holland et al., 1985), and Cushing's syndrome (Sonino et al., 1985; Loli et al., 1986; McCance et al., 1987; Engelhardt et al., 1989). From clinical data it has been supposed that the C17,20desmolase, the key enzyme of androgen biosynthesis, is most sensitive to the blocking effect of ketoconazole (Santen et al., 1983). To a lesser extent an inhibition of the 17a-hydroxylase, the 1 Ij-hydroxylase and the 18-hydroxylase has been postulated (De Coster et a[., 1987). While the site of action of ketoconazole with a relative selective inhibition of C17,20desmolase has been confirmed in rat testicular tissue (Lambert et al., 1986; Ayub & Levell, 1987; Vanden Bossche et al., 1990), the results of in-vitro studies with human adrenals remain conflicting. The aim of this study therefore was to investigate the invitro effect of ketoconazole on seven important steroidogenic enzyme systems in human adrenal tissue. Materials and methods Materials
The radiolabelled steroids 1,2,6,7-3H-progesterone (1 12 Ci/ mmol), 1,23H- 17a-hydroxyprogesterone (50 Ci/mmol), 1,23H-17a-hydroxypregnenolone (12 Ci/mmol), I , P H - I 1deoxycortisol(58.6 Ci/mmol), 4-'4C-corticosterone (50 mCi/ mmol), 4-14C-cortisol(55.5 mCi/mmol), 4-14C-1 1-deoxycorticosterone (58.5 mCi/mmol), 4J4C-1 1-deoxyeortisol(50-60 mCi/mmol), 4-14C-progesterone (50-60 mCi/mmol), 4-14C17a-hydroxyprogesterone (50-60 mCi/mmol) and 4J4Candrostenedione (52 mCi/mmol) were purchased from New England Nuclear, Boston, USA. 1,2-3H-corticosterone (49.7 Ci/mmol) was from Amersham Buchler, Braunschweig, FRG, all non-radioactive steroids were obtained from Sigma, Tauflcirchen, FRG. Thin-layer chromatography (TLC) plates Kieselgel 60 (F254) were obtained from Carl 163
164
D.Engelhardt et al.
319 HSD
Clinical Endocrinology (1991) 35
30 HSD 20a HSD
17 OH
110H CORTEOSTERONE
17,20 D
1
30 HSD
110H CORTISOL
18OH 18-OH-CORTICOSTERONE
1
A LD0STER0NE
Flg. 1 Main pathways of adrenal steroid biosynthesis. SCC denotes side-chain cleavage, HSD hydroxysteroid dehydrogenase, OH hydroxylase and D desmolase. Black bars indicate inhibition by ketoconazole.
Roth KG, Karlsruhe, FRG, ketoconazole (Nizoral) was purchased from Janssen GmbH Beerse, Belgium. Preparation and incubation of adrenal tissue
Human adrenal glands were obtained from 18 patients, who underwent adrenalectomy due to renal carcinoma (n= 13), renal transplantation (n= l), phaeochromocytoma (n= 3) or Cushing’s syndrome (n= 1). Adrenal tissue used for incubation experiments was macroscopically and histologically normal except in the patient with Cushing’s syndrome, who had adrenal hyperplasia. Immediately after aseptic removal, the adrenal glands were placed in ice-cold 0.9% NaCl solution and transferred to the laboratory. Slices of 100 mg wet weight were prepared at 4°C after the method of Deutsch (1936) and transferred to incubation vessels containing 2 ml of standard incubation mixture (Engelhardt et al., 1985). The mean & SEM protein content of 100 mg tissue slices, measured by the method of Lowry et al. (19Sl), was 10.9 k0.5 mg (n=9). After addition of radiolabelled precursors and various concentrations of ketoconazole (0-2000 p ~ ) the , adrenal tissue was incubated for 2 hours at 37°C in a shaking waterbath. The reaction was stopped by freezing at -40°C. All incubation experiments were done in triplicate. In preliminary experiments we found a two to three-fold stimulation of steroid biosynthesis by adrenal tissue after the addition of ACTH. Furthermore, the amounts of steroids
formed were related linearly to the amount of adrenal tissue, tissue protein and incubation time.
Analysis of steroid metabolism
The extraction and identification of tritium labelled metabolites were performed as described previously (Engelhardt et al., 1985). Before determination of steroids the tissue was homogenized mechanically and traces of 14C-labelledsteroids (10’ c.p.m.) were added for recovery calculations. After extraction with chloroform and distribution between methanol/n-heptane (2: 1 v/v), the labelled steroids and the metabolites were separated by thin-layer chromatography using the following systems, modified from the method of Neher (1964): chloroform/methanol (95 :5 v/v) and butylacetate/ ethylacetate/isoamylalcohol (1 : 1 : 1 by vol.) for incubation studies with )H-progesterone, )H-l 7a-hydroxyprogesterone and 3H-17a-hydroxypregnenolone;acetone/benzene (1 : 1 v/ v) and n-heptane/benzene/ethylacetate(1 : 1 :4 by vol.) for incubations with 11-deoxycortisol and corticosterone. If necessary, rechromatography was performed with the systems benzene/acetone (1 : 1 v/v) for separation of 17ahydroxyprogesterone, chloroform/methanol (95 :5 v/v) for separation of 16a-hydroxyprogesterone and I 1-deoxycorticosterone, chloroform/ethylacetate (8 :2 v/v) for separation of 17a,20a-dihydroxyprogesterone,androstenedione and
Ketoconazole and human adrenals
Clinical Endocrinology (1991) 35
progesterone. The labelled steroids on the thin-layer plates were localized by added non-labelled steroids. Quantitative analysis was made by liquid scintillation counting. C17,20desmolase activity was estimated by measuring the conversion of the added precursor 3H-17u-hydroxprogesterone (I 317 x mol) to 3H-17a,20a-dihydroxyprogesteroneand 3H-androstenedione. The 17a-hydroxylase activity was assessed by measuring the conversion of the added precursor mol) to 'H-17a-hydroxypro'H-progesterone (4-14 x gesterone, 16a-hydroxylase and 21-hydroxylase activity by measuring the amount of 3H-16u-hydroxyprogesterone and 'H-I 1-deoxycorticosterone in the same incubation experiments. Furthermore, the effect of ketoconazole on the followingenzyme activities was investigated: on 1ljl-hydroxylase by measuring the conversion of the precursor 3H-1I mol) to 'H-cortisol, on 18deoxycortisol (19-34x hydroxylase by measuring the transformation of added 3Hcorticosterone (20-22 x mol) to 'H-18-hydroxycorticosterone and on 21-hydroxylase by measuring the conversion of 3H-17a-hydroxyprogesterone (35-45 x mol) to 3H-lI-deoxycortisol.
165
I20
600
100
500
80
400
," 60
300
5-
+
u
0
p 4-
c
0 0
8 Ol
ES
40
200
20
I00
0,
?
2
I
I
I
1
0
Flg. 2 Effect of increasing concentrations of ketoconazole on A,
the amount of unmetabolized 3H-17~-hydroxyprogesterone; 0, formation of H- 17a,20a-dihydroxyprogesterone; and 0 , 'Handrostenedione after incubation of human adrenal tissue with 3H-17a-hydroxyprogesterone.100% control activity represents amounts of 83.8*8, 17.3+3 or 56.9&4 fmol per mg wet-weight and hour of incubation without ketoconazole. Values are means f SEM of three independent incubation experiments. NS, not significant;* PcO.05; ** P
