Tritium release from [19-3H]-19,19-difluoroandrost-4-ene-3,17-dione during inactivation of aromatase

Aromatase is a cytochrome P-450 enzyme involved in the conversion of androst-4-ene-3,17-dione to estrogen via sequential oxidations at the 19-methyl group. Previous studies from this laboratory showed that 19,19-difluoroandrost-4-ene-3,17-dione (5) is a mechanism-based inactivator of aromatase. The mechanism of inactivation was postulated to involve enzymic oxidation at, and hydrogen loss from, the 19-carbon. The deuteriated analogue 5b has now been synthesized and shown to inactivate aromatase at the same rate as the nondeuteriated parent (5). We conclude that C19-H bond cleavage is not the rate-limiting step in the overall inactivation process caused by 5. [19-3H]-19,19-Difluoroandrost-4-ene-3,17-dione (5b) with specific activity of 31 mCi/mmol was also synthesized to study the release of tritium into solution during the enzyme inactivation process. Incubation of [19-3H]19,19-difluoroandrost-4-ene-3,17-dione with human placental microsomal aromatase at differing protein concentrations resulted in time-dependent NADPH-dependent, and protein-dependent release of tritium. This tritium release is not observed in the presence of (19R)-10 beta-oxiranylestr-4-ene-3,17-dione, a powerful competitive inhibitor of aromatase. We conclude that aromatase attacks the 19-carbon of 19,19-difluoroandrost-4-ene-3,17-dione, as originally postulated.     

Digoxin-like immunoreactivity of 19-NOR and 19-OH-androst-4-ene-3,17-dione

Digoxin-like immunoreactivity of 19-OH-androst-4-ene-3,17-dione and 19-NOR-androst-4-ene-3,17-dione estimated by radioimmunoassay was by about four orders of magnitude lower than that of digoxin.     

The effects of in vivo administration of 10-propargylestr-4-ene-3,17-dione on rat ovarian aromatase and estrogen levels

We have previously demonstrated that 10-propargylestr-4-ene-3,17-dione (PED) functioned as an irreversible inhibitor of rat ovarian aromatase in vitro. These studies were undertaken to examine the in vivo effects of PED on rat ovarian aromatase activity and estrogen production. In the current experiments, a single injection of PED (0.5 or 2.5 mg/kg) was found to maximally inhibit aromatase at 3 h regardless of dose. Significant inhibition of enzyme activity by PED was observed beyond 18 h, although some recovery was noted at the lower dose (0.5 mg/kg). Concomitantly, ovarian estrogen levels were also maximally reduced at 3 h, however ovarian estrogen levels returned toward control values prior to the recovery in enzyme activity. Even though significant inhibition of enzyme activity was observed at 12 h following a single injection of PED, the effect of double injections of the inhibitor at 12 h intervals was surprisingly not cumulative. Similarly, continued multiple injections of PED revealed significant inhibition of enzyme activity and estrogen production several hours after the injection, but variations in effectiveness were observed by 12 h which changed in accordance with a circannual cycle in aromatase. Apparently other factors are involved with maintaining aromatase levels and compensating for reduced enzyme activity. These mechanisms are evidenced by a continuation of the rat reproductive cycle with prolonged PED administration and a reduced influence of PED in regard to enzyme inhibition at certain times of the year. Despite these variations in the duration of action of PED, no comparable changes were observed in effectiveness as an anti-tumor agent. These results suggest that complex mechanisms exist which regulate the activity of aromatase in order to maintain estrogen production. Further research using compounds such as PED may assist in elucidating the factors that modulate ovarian estrogen production.     

6-Substituted and 19-substituted androst-4-ene-3,17-dione derivatives as potential aromatase inhibitors [Poster 10]

Various 6-substituted and 19-substituted androst-4-ene-3, 17-dione analogs were synthesized and evaluated as aromatase inhibitors. The more effective competitive inhibitors were 6-azido, 6-thiocyanate, 6-propargyloxy, 6-(2'-azidoethyl)thio, 19-azido, 19-thiocyanato, and 19-methylxanthogenyl derivatives.     

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.     

The effect of the aromatase inhibitor, 4-(phenylthio)-4-androstene-3,17-dione, on dimethylbenz(A)anthracene-induced rat mammary tumors

4-(Phenylthio)-4-androstene-3,17-dione (4-PTAD), a known inhibitor of human placental aromatase, was examined as a growth inhibitor of DMBA-induced rat mammary tumors. Subcutaneous administration of 4-PTAD at dose levels of 25 or 50 mg/kg/day caused a significant decrease in hormone-dependent tumor growth. Resumption of tumor growth occurred when either the administration of inhibitor was stopped or when inhibitor was coadministered with estradiol indicating that suppression of tumor growth was due to inhibition of estrogen biosynthesis. Additionally, plasma levels of estradiol were found to be lower in the animals treated with 4-PTAD. The major metabolite of 4-PTAD in vitro was identified as 4-(phenylthio)-4-androstene-17 beta-ol-3-one and was found to have 60% of the aromatase inhibitory activity of 4-PTAD.     

Quantitative determination of 4-hydroxy-4-androstene-3,17-dione (4-OHA), a potent aromatase inhibitor, in human plasma, using isotope dilution mass spectrometry

An original method is described for the determination in human plasma of 4-hydroxy-4-androstene-3,17-dione (4-OHA), a potent aromatase inhibitor, by isotope dilution mass-spectrometry using 7,7-[2H2]-4-OHA as internal standard. This compound was synthesized starting from 7,7-[2H2]-4-androstene-3,17-dione. The procedure includes an extraction step using an Extrelut 1 column and a derivatization with N,o-bis(trimethylsilyl)trifluoroacetamide (BSTFA). The minimum detection level of the method is 0.650 pg and the coefficients of variation for the 0.5 ng/ml (plasma) and 5 ng/ml (plasma) concentrations are 3.2% (within assay) and 6.7% (between assay) and 1.86% (within assay) and 2.3% (between assay) respectively.     

Synthesis of 2,2-dimethyl-4-hydroxy-4-androstene-3,17-dione as an inhibitor of aromatase

2,2-Dimethyl-4-hydroxy-4-androstene-3,17-dione (4) has been synthesized and has been shown to be a powerful competitive inhibitor of aromatase (Ki = 11.4 nM). However, compound 4 does not cause time-dependent loss of enzyme activity, in contrast to the unmethylated parent compound, 4-OHA.     

Effects of 10-(2-propynyl)-estr-4-ene-3,17-dione (MDL 18,962)--a mechanism-based irreversible inhibitor of aromatase--in cultured human foreskin fibroblasts

In male subjects, peripheral aromatization of androgens accounts for most of the estrogen production, and skin is an important site of such enzymatic activity. We have studied the effects of a mechanism-based, irreversible aromatase inhibitor, 10-(2-propynyl)-estr-4-ene-3,17-dione (MDL 18,962) on androgen action and metabolism in cultured human foreskin fibroblasts. Cells were incubated simultaneously in the presence of substrate, androstenedione, and inhibitor, MDL 18,962. Aromatase activity was linear with time up to 3 h of incubation at 37 degrees C in the absence and presence of 1.0-10 nM inhibitor. The IC50 for four different cell strains ranged from 4.0 to 8.6 nM MDL 18,962. Kinetic analysis of competitive inhibition by the Eadie-Hofstee method yielded an apparent Ki of 2.75 nM for the inhibitor. Preincubation of cells with MDL 18,962 resulted in irreversible inhibition of aromatase activity which was time- and concentration-dependent. We calculated a Ki of 7.6 nM for MDL 18,962. Preincubation of cells with 25 nM MDL 18,962 suppressed enzyme activity for up to 6 h following removal of the inhibitor, before a return of enzyme activity due to synthesis of new enzyme. MDL 18,962 (0.2-20 microM) did not influence the 5 alpha-reduction of testosterone (200 nM). In addition, binding of dihydrotestosterone (2 nM) to androgen receptors was not affected by MDL 18,962 (25-1000 nM). In summary, MDL 18,962 is a specific, high potency inhibitor of aromatase. By virtue of its high binding affinity to the enzyme active site, it competes very effectively with substrate, resulting in irreversible inactivation of aromatase.     

Characterization of pregnant mare's serum gonadotropin-stimulated rat ovarian aromatase and its inhibition by 10-propargylestr-4-ene-3,17-dione

Aromatase, the important regulatory enzyme that converts androgens to estrogens, is found in relatively high levels in the human placenta. However, since the ovary is the major source of the estrogens in females, we undertook studies to compare the rodent ovarian enzyme with that from human placenta. Pregnant mare's serum gonadotropin (PMSG) markedly increases aromatase activity in the ovaries of immature rats, and this model was used in order to reproducibly obtain high enzyme levels. An injection of PMSG resulted in a specific stimulation of aromatase activity 12 times the increase in ovarian weight in 48 h. Kinetic studies demonstrated that, although the PMSG-stimulated ovarian microsomes had one-tenth the specific activity of the human placenta, the Km values were similar (about 33 and 44 nM, respectively). The potent inhibitor of placenta aromatase, 10-propargylestr-4-ene-3,17-dione, was used to further characterize the enzyme. It inhibited the rat aromatase with an I50 of 36 nM and exhibited time-dependent inhibition with a half-life of inactivation of 16 min and a Ki of 15 nM. These values are similar to those we obtained with the human enzyme (10 nM, 12 min, and 5 nM, respectively). The enzyme parameters in the presence and absence of the inhibitor suggest that the enzymes from the two sources are kinetically quite similar.     

Biological characterization of 10-(2-propynyl) estr-4-ene-3, 17-dione (MDL 18,962), an enzyme-activated inhibitor of aromatase

MDL 18,962 was shown to be a highly specific, potent (Ki = 3-4 nM), enzyme-activated inhibitor of aromatase with minimal intrinsic endocrine properties. The affinity of MDL 18,962 was higher for human and baboon placental aromatase than for rhesus placental or rodent ovarian aromatase. These species differences necessitated the development of a novel model of peripheral aromatase utilizing human enzyme. Human choriocarcinoma trophoblast xenografts in athymic nude mice were used for pharmacologic and pharmacokinetic evaluation of MDL 18,962. The ED50 for inhibition of aromatase activity in these trophoblast tumors at 6 h post-treatment was 1.4 mg/kg, s.c. and 3.0 mg/kg, oral. Preliminary results indicated that the ED50 for inhibition of peripheral aromatization of androgen by MDL 18,962 in female baboons was 0.01 mg/kg, i.v. and 4 mg/kg, oral.     

Behavioral action of estrogen in male hamsters: effect of the aromatase inhibitor, 1,4,6-androstatriene-3,17-dione (ATD)

This study examines three independent behavioral variables known to be activated by testosterone in the male hamster; namely, the tendency to approach the female, the tendency to leave the female, and the amount of sniffing directed to her. Both intact and testosterone-maintained castrated male hamsters were given the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) in subcutaneous, Silastic capsules. In intact males, there was an ATD dose-dependent increase in the tendency to leave the female and a decrease in the amount of olfactory investigation. The tendency of the male to approach the female was unaffected. The higher doses of ATD also abolished the ability of males to discriminate between females using vaginal odor cues. These results were confirmed in castrated males whose behavior was maintained at the intact level by testosterone implants. In addition, in both intact and castrated males, estradiol or diethylstilbestrol was able to reverse the behavioral changes induced by ATD. Our results indicate that estrogen produced by aromatization of testosterone activates behavior. We conclude that estrogen, by influencing some, but not all, components of masculine behavior, has a specific role in modulating male-female interactions.     

19-Norandrostenedione (4-estrene-3,17-dione) Inhibits Porcine Oocyte Maturation In Vitro

Recent work has shown that 19-norandrostenedione is a major steroidal component of porcine follicular fluid; however, little is known about its role(s) in the regulation of follicular function. This study was designed to examine the effect of 19-norandrostenedione on porcine oocyte maturation in vitro. Oocyte-cumulus complexes were isolated from medium (3–6-mm diameter)-sized prepubertal pig follicles and incubated for 12 h in medium with or without dibutyryl cyclic AMP ((Bu)₂cAMP, 1 mM) with or without testosterone (5 x 10^?7 M) or 19-norandrostenedione (5 x 10^?7 M). In medium alone, 70.8% of oocytes spontaneously resumed meiosis as evidenced by the occurrence of germinal vesicle breakdown. Oocyte maturation was inhibited by (Bu)₂cAMP (44.6% of oocytes matured). Although neither steroid alone affected maturation, both testosterone and 19-norandrostenedione enhanced the effect of (Bu)₂cAMP (22.5 and 19.6%, respectively, resumed meiosis). The effects of testosterone and 19-norandrostenedione on (Bu)₂cAMP-inhibited oocyte maturation were dose dependent and there was no significant difference between the actions of the steroids. The effect of 19-norandrostenedione was reversible and dependent on the presence of an intact cumulus. Hydroxyflutamide (SCH-16423), a nonsteroidal compound known to block androgen receptors, abolished the effects of both testosterone and 19-norandrostenedione on germinal vesicle breakdown, indicating that the actions of these steroids are truly androgenic. The results of this study suggest that 19-norandrostenedione may be of physiological importance in the regulation of porcine oocyte maturation.     

Effects of 4-hydroxy-4-androstene-3,17-dione and 10-propargylestr-4-ene-3,17-dione on the metabolism of androstenedione in human breast carcinoma and breast adipose tissues

The effects of 4-hydroxy-4-androstene-3,17-dione (4-OH-A) and 10-propargylestr-4-ene-3,17-dione (PED) on the aromatization of androstenedione (A) and the conversion of A to testosterone (T) were studied in incubations with breast carcinoma and breast adipose tissues. Parallel studies were carried out to determine the effects of 4-OH-A and PED on A metabolism in tissue from 5 patients with breast carcinoma. At 11 μM, both compounds fully inhibited aromatization, whereas the conversion of A to T was decreased in only 2 incubations.Studies with varying concentrations of 4-OH-A and PED demonstrated that both compounds inhibited estrone (E₁) formation by 80% at a concentration of 0.085 μM, with maximum effect at 0.34 μM. 90% inhibition of estradiol (E₂) formation was observed at inhibitor concentrations of 0.17 μM or greater. T formation was slightly affected at 0.67 μM, but was progressively inhibited with increasing 4-OH-A or PED concentrations, reaching 70% at 11 μM.Similar experiments with 4-OH-A in breast adipose tissue homogenates showed that a concentration of 0.1 μM was sufficient to inhibit aromatization while T inhibition required 11 μM.4-OH-A and PED are selective inhibitors of aromatization in human breast tissues and may provide a mechanism for controlling estrogen responsive processes.     

Effect of 1,4,6-androstatriene-3,17-dione (ATD), 4-hydroxy-4-androstene-3,17-dione (4-OH-A) and 4-acetoxy-4-androstene-3,17-dione (4-Ac-A) on the 5 alpha-reduction of androgens in the rat prostate

The present study reports the effects exerted by 1,4,6-androstatriene-3,17-dione (ATD), 4-hydroxy-4-androstene-3,17-dione (4-OH-A) and 4-acetoxy-4-androstene-3,17-dione (4-Ac-A), three steroids known to inhibit the aromatization of androgens to estrogens, on the in vitro metabolism of labelled testosterone (T), dihydrotestosterone (DHT) and androstenedione (delta-4-A) in the ventral prostate of adult male rats. It has been found that ATD, in the concentration tested, does not influence the conversion of labelled T into DHT, but decreases the formation of 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol (diols). On the contrary, 4-OH-A and 4-Ac-A simultaneously decrease the formation of DHT and the diols. When T is used as the substrate, the presence in the medium of these three steroids enhances the formation of delta-4-A and of 5 alpha-androstanedione (5 alpha-A). ATD, but not 4-OH-A and 4-Ac-A inhibits the conversion of labelled DHT into the diols. The transformation of labelled delta-4-A into 5 alpha-A is not modified by either ATD or 4-OH-A, while 4-Ac-A exerts only a small inhibition. These results suggest that the three aromatase inhibitors tested are able to profoundly modify the metabolism of T in the ventral prostate of the rat. In particular: 4-OH-A and 4-Ac-A are able to inhibit the conversion of T into DHT; ATD is able to inhibit the conversion of DHT into the diols; ATD and 4-OH-A do not inhibit the process of 5 alpha-reduction of delta-4-A into 5 alpha-A, while 4-Ac-A exerts only a minor effect. It is suggested that in the ventral prostate of the rat there are two different 5 alpha-reductase isoenzymes, one sensitive to the inhibitory effect of the steroid tested and which is responsible for the conversion of T into the 5 alpha-reduced metabolites of the 17-OH series (DHT and the diols), and a second one, insensitive to the effects of the three steroids, which affects the conversion of delta-4-A into 5 alpha-A.     

Androst-5-ene-7,17-dione: a novel class of suicide substrate of aromatase.

5-En-7-one steroid 1 was found to be a potent inhibitor of aromatase. This along with its 19-hydroxy derivative 7 was characterized as suicide substrate of human placental aromatase (k(inact)'s of 0.069 and 0.058 min-1 and KI's of 143 nM and 11.1 microM, respectively, for steroids 1 and 7). The results suggest that the 19-oxygenation would be involved in the irreversible inactivation of aromatase by the 5-en-7-one steroids.