D. Beerens

R 76713, a new specific non-steroidal aromatase inhibitor

The effects of R 76713, a new triazole derivative, on rat ovarian, testicular and adrenal steroidogenesis were investigated both in vitro and in vivo. In vitro R 76713 is a very potent inhibitor of the aromatase enzyme in rat granulosa cells, showing an IC50-value of 3.0 +/- 0.2 nM. The compound is about 1000 times more active than aminoglutethimide which shows an IC50-value of 3900 +/- 2800 nM in the same system. R 76713 is also a highly selective aromatase inhibitor. In cultures of ovarian, testicular and adrenal cells, formation of progesterone, androgens and glucocorticoids was only affected by drug concentrations higher than 1 microM. In vivo, single oral drug doses of 0.05 mg/kg lowered plasma estradiol levels of PMSG-primed female rats by more than 90%. An ED50-value of 0.005 mg/kg could be calculated. A single oral dose of 1 mg/kg suppressed plasma estradiol levels almost completely for 24 h. A dose of 0.1 mg/kg lowered plasma estradiol by more than 90% for 8 h. In vivo, R 76713 also showed a highly selective profile. In LHRH/ACTH-injected rats, plasma levels of testicular and adrenal steroids remained unchanged after administration of a drug dose of 20 mg/kg. R 76713 at drug concentrations of 10 microM, showed no interaction in vitro with estrogen-, progestin-, androgen- and glucocorticoid-receptors. Given orally at 20 mg/kg for 3 days the compound also showed no estrogen or androgen agonistic or antagonistic effects.

EFFECTS OF HIGH DOSE KETOCONAZOLE THERAPY ON THE MAIN PLASMA TESTICULAR AND ADRENAL STEROIDS IN PREVIOUSLY UNTREATED PROSTATIC CANCER PATIENTS

In vitro, ketoconazole has been shown to block testicular and adrenal 17,20‐lyase, which converts progestins to androgens. At higher concentrations, it also inhibits 11β‐hydroxylase, 20,22‐desmolase and 17α‐hydroxylase. To determine the differential hormonal effects of a 2‐week ketoconazole high‐dose therapy, the plasma levels of 10 major androgens, gluco‐ and mineralocorticoids were measured in 14 previously untreated patients with metastatic prostate cancer. Within 24 h, plasma testosterone fell from 14·6 ± 1·4 nmol/l (mean ± SEM) to 3·7 ± 0·7 nmol/l. Thereafter, it decreased to about 2·5 nmol/l and remained at that level. Plasma androstenedione and dehydroepiandrosterone decreased more gradually, respectively from 3·1 ± 0·4 nmol/l to 0·64 ± 0·17 nmol/l and from 6·6 ± 1·0 nmol/l to 2·82 ± 0·55 nmol/l (on day 14). In contrast, 17α‐hydroxyprogesterone and progesterone rose respectively 2‐ and 5‐fold. Plasma cortisol and aldosterone levels remained unchanged whereas 11‐deoxycorticosterone and 11‐deoxycortisol rose by factors of 14 and 6·7 respectively. Plasma corticosterone also increased, but to a much lesser extent (3‐fold). These results demonstrate that ketoconazole high dose therapy blocks mainly the 17,20‐lyase of both adrenal and testis. In addition it inhibits mitochondrial 11β‐hydroxylase to a lesser extent. The inhibition of 20,22‐desmolase also seems to be of little clinical relevance. However, since clinical or laboratory symptoms suggestive of hypo‐adrenalism have been reported in a small minority of patients, replacement therapy should be considered in such cases.

Potency and selectivity of the aromatase inhibitor R 76 713. A study in human ovarian, adipose stromal, testicular and adrenal cells

The effects of R 76,713 on steroidogenesis were studied in primary cultures of four different human cell types, i.e. ovarian granulosa cells, adipose stromal cells, testicular cells and adrenal cells. In human granulosa cells aromatization of [1 beta, 2 beta-3H]androstenedione (as measured by the release of tritiated water) showed a Km (Michaelis constant) of 78 nM. R 76,713 competitively inhibited aromatization with a Ki (dissociation constant of the enzyme-inhibitor complex) of 1.6 nM. In human adipose stromal cells aromatization was measured by following the conversion of androstenedione to estrone and 17 beta-estradiol. In this system a Km for aromatization of androstenedione of 10.8 nM was found. R 76,713 again showed competitive kinetics with a Ki-value of 0.14 nM. In human testicular cells the synthesis of the androgens testosterone, androstenedione and dehydroepiandrosterone was only inhibited by drug concentrations exceeding 10(-6) M. At 10(-5) M of R 76,713, steroid concentrations were lowered to 56, 64 and 81% of the control for testosterone, androstenedione and dehydroepiandrosterone respectively. Concomitantly, a slight increase in the levels of pregnenolone (138% of the control) and progesterone (133% of the control) was seen. In human adrenal cells the synthesis of cortisol and aldosterone was slightly affected by R 76,713 also at concentrations exceeding 10(-6) M. At 10(-5) M of R 76,713 the concentrations of cortisol and aldosterone were lowered to respectively 59 and 51% of the control. At the same drug concentration the precursors 11-deoxycortisol and 11-deoxycorticosterone rose to 189 and 147% of the control. These results show that in primary cultures of human cells, R 76,713 is a very potent aromatase inhibitor with a selectivity of at least 1000-fold compared to other steps in steroidogenesis.

Aromatase inhibition by the antifungal ketoconazole.

The aromatase inhibitory properties of the antifungal ketoconazole were compared with those of aminoglutethimide. In rat granulosa cells ketoconazole and aminoglutethimide showed IC50 values for aromatase inhibition of 2 X 10(-6) and 6 X 10(-7) M respectively. In the rat, in vivo, ketoconazole was 5 times less potent than aminoglutethimide. In young women, 400 mg of ketoconazole only marginally lowered plasma levels of estradiol-17 beta. It is concluded that ketoconazole is not a compound of choice for clinical use as an aromatase inhibitor.

Heteroaryl urea inhibitors of fatty acid amide hydrolase: Structure-mutagenicity relationships for arylamine metabolites

The structure-activity relationships for a series of heteroaryl urea inhibitors of fatty acid amide hydrolase (FAAH) are described. Members of this class of inhibitors have been shown to inactivate FAAH by covalent modification of an active site serine with subsequent release of an aromatic amine from the urea electrophile. Systematic Ames II testing guided the optimization of urea substituents by defining the structure-mutagenicity relationships for the released aromatic amine metabolites. Potent FAAH inhibitors were identified having heteroaryl amine leaving groups that were non-mutagenic in the Ames II assay.

COMPARATIVE EFFECTS OF KETOCONAZOLE ON RAT, DOG AND HUMAN TESTICULAR STEROIDOGENESIS

Ketoconazole is an antifungal azole derivative which also inhibits the cytochrome P-450(17)alpha, catalyzing the conversion of progestins into androgens. The effects of ketoconazole on human, dog and rat testosterone biosynthesis were compared using short term incubations of dispersed testicular cells. The results showed that ketoconazole inhibited androgen biosynthesis at lower concentrations in dispersed human testicular cells (IC50: 0.08 mumol/l) than in canine (IC50: 0.1 mumol/l) and rat cells (IC50 greater than or equal to 0.2 mumol/l). Furthermore, they demonstrated that ketoconazole first inhibited the 17,20-lyase activity and then the 17-hydroxylation in rat and dog cells whereas only the 17-hydroxylation was affected in human cells.

Effects of etomidate on Cortisol biosynthesis in isolated guinea-pig adrenal cells: comparison with metyrapone

The effects of the iv hypnotic etomidate on Cortisol biosynthesis have been investigated in short term incubations of dispersed guinea-pig adrenal cells and were compared with those produced by metyrapone. Fifty per cent inhibition of Cortisol output was obtained at a final medium concentration of 3.5 10−8 M (basal), 2.8 10−8 M (ACTH-stimulated) for etomidate and of 5.107 M (stimulated) for metyrapone. In the presence of etomidate, 11-deoxycortisol at 5.10−8 M reached a peak value of 244 ± 11% of control (mean ± SE, n = 7). 17α-Hydroxyprogesterone and progesterone were not significantly affected up to 10−7 M, but at higher concentrations, all three precursors fell under their control values. Metyrapone induced a progressive rise of 11-deoxycortisol, from 10−7 M upwards, to a maximum level at 10−5 M (210 ± 15 % of control, mean ± SE, n = 5). 17-Hydroxyprogesterone and progesterone concentrations were not significantly modified by metyrapone. The less active hypnotic L-enantiomer of etomidate had almost no inhibitory effect on Cortisol production. The results obtained so far suggest that etomidate is a potent inhibitor of the mitochondrial cytochrome P-450 enzymes of the adrenal cortex, mainly the 11 gB-hydroxylase. At higher dose the cholesterol side-chain cleavage enzyme system seemed also to be affected.

Vergleich der Wirkungen von Ketoconazol auf die testikuläre Steroidogenese bei Ratte, Hund und Mensch

Ketoconazol ist ein oral wirksames Breitband-Anti-mykotikum. Bei niedrigen Konzentrationen (nM) hemmt dieser Stoff die Ergosterinsynthese bei Hefen und Pilzen. Bei hoheren Konzentrationen (0,1 bis 10 μM) hemmt dieses Imidazolderivat bei Saugetieren mehrere zytochrom-P-450-abhangige Enzyme, vor allem aber solche, die bei der Steroidogenese eine Rolle spielen [3]. Diese Ergebnisse haben zu weiteren Studien angeregt, die auf die Bedeutung hoher Ketoconazol-Dosen (d.h. 400mg dreimal taglich) bei der First- und Second-line-Behandlung von Patienten mit Prostatakrebs hinwiesen (Ubersichten s. [1, 2]).