FCE 28260, a new 5 alpha-reductase inhibitor: in vitro and in vivo effects.

FCE 28260 is a novel inhibitor of 5 alpha-reductase (5 alpha R), the enzyme responsible for the conversion of testosterone (T) to 5 alpha-dihydrotestosterone (DHT). The compound caused inhibition of rat and human prostatic enzymes, with IC50 values of 15 and 16 nM, respectively, compared to the values of 30 and 52 nM shown by finasteride. Furthermore, FCE 28260 was highly potent in inhibiting human recombinant 5 alpha R type 2 and 1 isozymes, showing IC50 values of 3.3 and 36 nM, and therefore it was more potent than finasteride (IC50 values of 8.5 and 470 nM) on both isozymes. In prepubertal, T-implanted castrated rats, FCE 28260, given orally for 7 days, reduced ventral prostate growth with an ED50 of 0.8 mg/kg, i.e. five times lower than that shown by finasteride. No anti-androgenic activity in DHT-implanted castrated rats was found up to 10 mg/kg/day. In adult male rats, FCE 28260 reduced prostatic DHT concentrations 6 h after oral dosing with a potency similar to that of finasteride (65% reduction at 1 mg/kg) but was found to be markedly more potent than the reference compound at 24 h (74% reduction in prostate DHT at 10 mg/kg, compared to 26% reduction induced by finasteride). These results indicate that FCE 28260 represents a marked improvement over finasteride.

Effects of 5 alpha-reductase inhibitors on intraprostatic androgens in the rat.

FCE 27837 is a novel inhibitor of 5 alpha-reductase, the enzyme responsible for the conversion of testosterone (T) to 5 alpha-dihydrotestosterone (DHT). The compound caused inhibition of human and rat prostatic enzymes, with IC50 values of 51 and 60 nM, respectively. The in vivo effect of FCE 27837 on 5 alpha-reductase was evaluated in adult male rats, treated orally at 10 mg/kg/day for 10 days. The compound caused 33 and 42% reductions in ventral prostate and seminal vesicle weights, respectively. The prostatic content of DHT, measured 6 h after the 10th dose of FCE 27837, was reduced by 75%, whereas T content increased by 442%. Similar effects were observed with 10 mg/kg/day of finasteride, whereas epristeride, tested at the same oral dose, was found to be the least effective compound, decreasing prostate weight by 22% and DHT content by 46%. Castration caused > 90% reductions in prostatic weight and prostatic DHT.

Novel aromatase and 5 alpha-reductase inhibitors.

Inhibitors of aromatase and 5 alpha-reductase may be of use for the therapy of postmenopausal breast cancer and benign prostatic hyperplasia, respectively. FCE 27993 is a novel steroidal irreversible aromatase inhibitor structurally related to exemestane (FCE 24304). The compound was found to be a very potent competitive inhibitor of human placental aromatase, with a Ki of 7.2 nM (4.3 nM for exemestane). In preincubation studies with placental aromatase FCE 27993, like exemestane, was found to cause time-dependent inhibition with a higher rate of inactivation (t1/2 4.5 vs 15.1 min) and a similar Ki(inact) (56 vs 66 nM). The compound was found to have a very low binding affinity to the androgen receptor (RBA 0.09% of dihydrotestosterone) and, in contrast to exemestane, no androgenic activity up to 100 mg/kg/day s.c. in immature castrated rats. Among a series of novel 4-azasteroids with fluoro-substituted-17 beta-amidic side chains, three compounds, namely FCE 28260, FCE 28175 and FCE 27837, were identified as potent in vitro and in vivo inhibitors of prostatic 5 alpha-reductase. Their IC50 values were found to be 16, 38 and 51 nM for the inhibition of the human enzyme, and 15, 20 and 60 nM for the inhibition of the rat enzyme, respectively. When given orally for 7 days in castrated and testosterone (Silastic implants) supplemented rats, the new compounds were very effective in reducing prostate growth. At a dose of 0.3 mg/kg/day inhibitions of 42, 36 and 41% were caused by FCE 28260, FCE 28175 and FCE 27837, respectively.

Endocrine properties of the testosterone 5 alpha-reductase inhibitor turosteride (FCE 26073).

Turosteride was tested in a series of studies for its effect on 5 alpha-reductase and for its possible influence on other steroidogenic enzymes and on steroid receptors. The compound was found to inhibit human and rat prostatic 5 alpha-reductases with IC50 values of 55 and 53 nM, respectively, whereas it caused a less marked inhibition of the dog enzyme (IC50 2.2 microM). Turosteride showed no relevant effect on rat adrenal C20,22-desmolase (IC50 254 microM) and human placental aromatase (IC50 > 100 microM), and only at relatively high concentrations it caused inhibition of human placental 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-I) (IC50 2.5 microM). Turosteride was found to be a selective 5 alpha-reductase inhibitor showing no noteworthy binding to receptors for androgens (relative binding affinity, RBA, 0.004%), estrogens (< or = 0.005%), progesterone (< 0.005%), glucocorticoids (< 0.01%) and mineralocorticoids (< 0.03%). Its biochemical profile was similar to that of finasteride, whereas 4-MA (17 beta-N,N-diethyl-carbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one) was confirmed to be a non-selective 5 alpha-reductase inhibitor, showing a degree of binding affinity to the androgen receptor (RBA 0.1%) and a marked inhibition of 3 beta-HSD-I (IC50 32 nM). When given orally in immature castrated rats together with subcutaneous testosterone propionate (TP) for 7 consecutive days, turosteride reduced the ventral prostate and seminal vesicle growth promoting effect of TP, with IC50 values of approximately 5 and 6.7 mg/kg/day, whereas levator ani weight was unchanged. In comparison, 4-MA was approx. 3-fold less potent than turosteride in reducing the prostate and seminal vesicle weights and caused a marked reduction of levator ani weight, thus showing its unselectivity.

Synthesis and aromatase inhibition by potential metabolites of exemestane (6-methylenandrosta-1,4-diene-3,17-dione).

Exemestane (6-methylenandrosta-1,4-diene-3,17-dione; FCE 24304) is an orally active irreversible aromatase inhibitor which is in phase II clinical evaluation for the potential therapy of postmenopausal breast cancer. A series of exemestane analogs, with modifications at the 6-methylene group and with additional reduction at the 17-keto group, were synthesized as potential metabolites and tested in vitro for their effect on human placental aromatase. All these new analogs were found to be less potent in inhibiting aromatase than exemestane. The most effective compound was the 17 beta-hydroxy-derivative (compound 2), which is 2.6-fold less potent than exemestane [50% inhibitory concentration (IC50) 69 and 27 nM, respectively]. The various C-6 modified derivatives of the 17-oxo series were found to inhibit the aromatase enzyme in the following descending order: 6-methylene (exemestane) > 6-spirooxirane (6) > 6 beta-hydroxymethyl (11) > 6-hydroxymethyl (7) > 6 beta-carboxy (13), showing IC50 values of 27, 206, 295, 2,300, and 7,200 nM, respectively. The 17 beta-hydroxy analogs of some of the above mentioned compounds were also synthesized (3,4,12) and found to be 3-8-fold less potent than the corresponding 17-keto analogs.

Hormonal effects of turosteride, a 5 alpha-reductase inhibitor, in the rat.

Turosteride [FCE 26073; 1-(4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carbonyl)-1,3- diisopropylurea] is a novel inhibitor of 5 alpha-reductase, the enzyme responsible for the conversion of testosterone (T) to 5 alpha-dihydrotestosterone (DHT). The compound caused inhibition of rat and human prostatic enzymes, with IC50 values of 55 and 53 nM, respectively. In addition, turosteride did not show any relevant binding affinity to the rat prostate androgen receptor (IC50 84 microM; relative binding affinity 0.004% of DHT). The endocrine effects of turosteride were evaluated in adult male rats, treated orally at daily doses of 3, 10 and 30 mg/kg for 20 days. At these doses, the compound reduced the ventral prostate weight by 10, 33 and 42% and the intraprostatic total DHT content by 61, 74 and 78%, respectively, whereas no change in the intraprostatic content of T was observed. Turosteride caused a 40% reduction of serum DHT levels which, however, did not reach statistical significance, whereas serum T levels were similar to control animals. No effect on serum luteinizing hormone or prolactin was observed. These results indicate that the antiprostatic effect of turosteride in the adult rat is related to inhibition of the conversion of T to DHT. However, at variance with other 5 alpha-reductase inhibitors (e.g. finasteride), turosteride caused a decrease in prostatic DHT not associated with a secondary increase in T content. This peculiarity of turosteride may represent an improvement of the compound over other inhibitors.

17 beta-acylurea derivatives of 4-azasteroids as inhibitors of testosterone 5 alpha-reductase.

A series of 17 beta-acylurea-4-aza-5 alpha-androstan-3-one derivatives has been assayed in vitro as inhibitors of testosterone 5 alpha-reductase, using the particulate fraction of human hyperplastic prostate and rat prostate as enzyme sources. The most active derivatives were 1-[4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carbonyl]- 1,3-dicyclohexylurea (compound 1) and 1-[4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carbonyl]- 1,3-diisopropylurea (compound 3) which demonstrated IC50 values of 41 and 55 nM for the human enzyme and of 83 and 53 nM for the rat enzyme, respectively. Neither compound showed any relevant binding affinity to the rat prostate androgen receptor (IC50 of approximately 100 and 84 microM). When given orally in immature castrated rats together with subcutaneous testosterone propionate (TP) for 7 consecutive days, compound 3 (laboratory code FCE 26073), at 3 mg/kg/day, significantly decreased the ventral prostate growth promoting effect of TP by 40-50%, whereas compound 1 was ineffective up to the dose of 10 mg/kg/day.

4-Aminoandrostenedione derivatives: a novel class of irreversible aromatase inhibitors. Comparison with FCE 24304 and 4-hydroxyandrostenedione.

FCE 24928 (4-aminoandrosta-1,4,6-triene-3,17-dione) was selected among a series of 4-aminoandrostenedione derivatives as a novel irreversible aromatase inhibitor. Its in vitro and in vivo properties have been studied and compared to FCE 24304 (6-methylenandrosta-1,4-diene-3,17-dione) and 4-OHA (4-hydroxyandrostenedione). FCE 24928 caused time-dependent inhibition of human placental aromatase with a t1/2 of 4 min and Ki of 59 nM. Enzyme inactivation by FCE 24928 was faster than by FCE 24304 (t1/2 13.9 min). In PMSG-treated rats, microsomal ovarian aromatase activity was reduced 24 h after FCE 24928 dosing by both the s.c. (ED50 1.2 mg/kg) and the oral (ED50 14.1 mg/kg) routes. The compound was more potent than FCE 24304 and 4-OHA (ED50 1.8 and 3.1 mg/kg s.c.). FCE 24928 did not show any interference with 5 alpha-reductase and desmolase activity nor any significant binding affinity for androgen and estrogen receptors. Slight binding affinity for androgen receptor was observed with FCE 24304 and 4-OHA (0.21 and 0.25% of DHT). In immature, castrated rats, FCE 24928 did not show any intrinsic androgenic activity, up to 100 mg/kg/day s.c., in contrast to a slight androgenic activity observed with FCE 24304 at 10 mg/kg s.c.

Aromatase inhibition and experimental antitumor activity of FCE 24304, MDL 18962 and SH 489.

Human placental aromatase inhibitory properties of FCE 24304, MDL 18962, SH 489 and 4-hydroxyandrostenedione (4-OHA) were compared. The compounds caused time-dependent enzyme inactivation with t1/2 values of 13.9, 13.1, 45.3 and 2.1 min and Ki values of 26.0, 0.7, 2.0 and 29.0 nM respectively. The antitumor activity of FCE 24304, MDL 18962 and SH 489 was studied on the DMBA-induced mammary tumor in rats, at daily s.c. doses of 10 and 50 mg/kg. FCE 24304 induced 30 and 73% regressions of established tumors, associated with 86 and 93% decrease in total ovarian aromatase activity. SH 489 and MDL 18962 did not affect tumor growth. FCE 24304, like 4-OHA, was shown to inhibit LH hypersection in castrated rats. A gonadotropin suppressive effect could contribute to the antitumor activity of aromatase inhibitors in intact DMBA-induced tumor bearing rats.

6-Methylenandrosta-1,4-diene-3,17-dione (FCE 24304): a new irreversible aromatase inhibitor.

FCE 24304 (6-methylenandrosta-1,4-diene-3,17-dione), a new irreversible aromatase inhibitor, has been identified and characterized in vitro and in vivo. The compound caused time-dependent inactivation of human placental aromatase with a t1/2 of 13.9 min and ki of 26 nM. When tested in PMSG-treated rats, ovarian aromatase activity was reduced 24 h after dosing by both the s.c. (ED50 1.8 mg/kg) and the oral (ED50 3.7 mg/kg) routes. No interference with 5 alpha-reductase activity nor any significant binding affinity for estrogen receptor was found. Slight binding affinity for the androgen receptor (RBA 0.2% of DHT) was observed.

Antinociceptive, prolactin releasing and intestinal motility inhibiting activities of dermorphin and analogues after subcutaneous administration in the rat.

A series of analogues and shorter homologues of dermorphin (DM), a frog skin heptapeptide with potent morphine-like activity, have been assayed in the rat after subcutaneous (SC) administration at the screening dose of 4 mg/kg. The effects taken into account are: analgesia (tail-pinch test), stimulation of prolactin (PRL) secretion, and inhibition of gastro-intestinal (GI) motility (charcoal meal transit). Effective doses were calculated for the most active compounds. The potency of DM (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) in the different tests was: tail-pinch: ED50 = 0.83 mg/kg; PRL release: ED100 = 0.3 mg/kg; inhibition of GI motility: ED30 = 1.8 mg/kg.

Effects of contraceptive agents on drug metabolism in various animal species.

The effect on liver microsomal enzyme activity of three steroid contraceptive drug (SCD) combinations was compared in rats, mice and guinea-pigs. Lynestrenol plus mestranol, norethisterone plus mestranol and norethynodrel plus mestranol were given orally for 4 consecutive days (acute treatment) or 30 days (chronic treatment) at various doses eliciting an experimentally controlled antifertility activity which varied in its extent. In rats and mice all the combined treatments (with the exception of norethynodrel plus mestranol in mice) were active as inducers of liver microsomal enzymes. This induction seems to be mediated mainly by the progestogenic compounds. Oestrogens showed a very poor effect bordering on significance only in a few cases. No effect on liver microsomal protein or cytochrome P 450 concentration was obtained after treatment with doses capable of increasing the microsomal enzyme activity. The activity of the liver microsomal enzymes did not appear to be reduced immediately (2 h) after the last administration of the SCD given during 4 or 30 days. Contraceptive treatments at doses capable of eliciting complete antifertility activity were inactive on liver microsomal enzyme activity in guinea-pigs.