Prolame produces anxiolytic- and antidepressant-like effects in middle-aged female rats with less uterotrophic effects than 17ß-estradiol.

Estrogen hormone replacement therapy (EHRT), improving women's life quality at menopause, reduces anxiety and depression symptoms associated with ovarian hormonal decline. However, its potential adverse effects, like thromboembolism and cancer risk, limit its use. Prolame is a synthetic 17ß-amino estrogen with antithrombotic actions that exerts anxiolytic- and antidepressant-like effects on young adult ovariectomized female rats. It is unknown if prolame's effects may be observed in age and endocrine conditions emulating menopause. This study aimed to identify the antidepressant- and anxiolytic-like effects of prolame and E2 (used as a reference estrogen treatment) in middle-aged female rats coursing with irregular cycles, in two different conditions: ovariectomized or gonadally intact. Results were compared with those from young adult ovariectomized rats. Prolame (60 or 120 µg/kg), 17ß-estradiol (E2, 40 or 80 µg/kg), or vehicle were chronically administered, and their effects were evaluated in the elevated plus-maze, defensive burying behavior test, open field test, and forced swimming test. Uterotrophic actions were estimated by uterine weight related to body weight. Prolame and E2 produced robust anxiolytic- and antidepressant-like effects in young adult ovariectomized rats, but these effects were absent in gonadally intact middle-aged rats. Interestingly, only prolame induced anxiolytic- and antidepressant-like effects in middle-aged ovariectomized rats. Uterotrophic effects of prolame were weaker than E2 effects, notably in middle-aged females. Altogether, present data support the notion that prolame has the potential to be considered an EHRT with relevant psychoactive actions and with apparently lower adverse-side effects, especially in middle-aged populations.

Differential effect of the 17ß-aminoestrogens prolame, butolame and pentolame in anxiety and depression models in rats.

Estrogens of clinical use produce consistent antidepressant- and anxiolytic-like effects in animal models of menopause. Regulation of the hypothalamic-pituitary-adrenal (HPA) or stress axis, has been proposed as a pathway through which estrogens improve affective-like behaviors. Anticoagulant 17ß-aminoestrogens (17ß-AEs) butolame and pentolame mimic some effects of estradiol (E2), i.e., on female rodent sexual behavior, with opposite actions on coagulation. However, their psychoactive actions have not been explored. On the basis of similitude with E2's effects, we hypothesized that these 17ß-AEs would induce anxiolytic- and antidepressant-like effects, which would be reflected in a reduction of activity in the HPA axis. In ovariectomized female rats, chronic treatment with prolame (60 µg/kg), butolame (65 µg/kg) and pentolame (70 µg/kg) reduced anxiety-like behavior in the elevated plus maze (evidenced by an increase in time in open arms, E2 (40 µg/kg) +176%; prolame +201%; butolame, +237%; and pentolame +295%, in comparison to the control vehicle group 100%). Pentolame also decreased significantly anxiety-like behavior in the burying behavior test. Prolame and E2 produced a significantly antidepressant-like action, which was not induced by butolame and pentolame. Behavioral effects of 17ß-AEs (and E2) on anxiety and depression did not follow the same pattern than corticosterone or E2 levels; they also were associated to changes in locomotor activity, evaluated by the open field test. These results constitute the first evidence of specific and selective actions of butolame and pentolame as anxiolytics for females with a hypoestrogenic condition. Results also confirm the potential of prolame as an antidepressant steroid with equivalent actions to E2. Psychoactive properties of 17ß-AEs in combinations with reduced adverse effects on coagulation, suggest that 17ß-AEs may be a good alternative replacement therapy for women with symptoms associated with menopause.

Participation of estrogen receptors in the antidepressant-like effect of prolame on the forced swimming test.

Estrogen therapy may produce antidepressant-like actions, but the side effects, such as thromboembolic events, may restrict its use among women. The 17ß-aminoestrogens (AEs) [prolame [17ß-(3-hidroxy-1-propylamino)-1,3,5(10)-estratrien-3-ol)], butolame [17ß-(3-hidroxy-1-butylamino)-1,3,5(10)-estratrien-3-ol)], and pentolame [17ß-(5-hidroxy-1-pentylamino)-1,3,5(10)-estratrien-3-ol)] induce estrogenic and anticoagulant actions, effects that could prove advantageous in an estrogen therapy; however, their antidepressant-like effects have not been described. The objective of this study was to determine the effect of these 17ß-AEs (prolame, butolame and pentolame) in the forced swimming test (FST), an animal model sensitive to antidepressant drugs, and to establish the role of estrogen receptors in such actions. Ovariectomized female rats treated with prolame (10-200 µg/rat) showed a reduction in immobility and an increase in active behaviors in the FST, while this effect was not produced by butolame and pentolame (10-200 µg/rat). The antidepressant-like effect of prolame was similar to that of 17ß-estradiol (E2, 5-20 µg/rat), sharing with it a biphasic profile but at higher doses. Antidepressant-like actions of prolame and E2 were not associated with changes in locomotor activity. With respect to a control group tamoxifen (15 mg/kg) by itself produced no changes in all behavioral evaluations, but canceled the antidepressant-like effect of prolame and E2. It is concluded that estrogen receptors participate in antidepressant-like effect of both estrogens in the FST. Antidepressant-like activity of different AEs is discussed considering their differences in chemical structure and the schedule used. Our results show additional central actions of prolame besides its pro-sexual, anti-coagulant, estrogenic and anxiolytic activity.

Effects of 17beta-aminoestrogens on the sexual behavior of female rats.

17beta-aminoestrogens (AEs) produce anticoagulant effects in rats contrastingwith 17beta-estradiol (E2) procoagulant effects, their estrogenic effects are similar to E2, decreasing serum luteinizing hormone (LH), increasing uterine weight (Uw), activate transcription through the ERalpha and ERbeta receptors and pentolame induces progesterone (P) receptors in the anterior pituitary of ovariectomized (Ovx) rats similarly to E2, suggesting possible effects on female rats' sexual behavior. This work evaluated the AEs prolame, butolame, pentolame compared to E2 and estradiol benzoate (EB) as facilitators on the rat lordotic behavior. Dose-response curves were performed in rats by single subcutaneous (s.c.) injection (timezero) of: E2 (approximately 0.3, 3, 30, 60, 300 microg/kg); EB (approximately 0.4, 4, 40, 80, 400 microg/kg); prolame, butolame, pentolame (approximately 40, 400, 2000 or 4000 mg/kg), vehicle (corn oil; 300 microL/day; approximately 1.2 mL/kg) as control; 24 h after, P (1 mg/rat in 100 microL of corn oil; approximately 4 to 5 mg/kg) was administered, and 5 to 7 h later LQ was evaluated (number of lordosis displays/number of mounts x 100). E2, EB and AEs followed by P administration, induced lordosis in a dose-dependent manner. Prolame induced an LQEmax of 92, butolame85, EB 81, pentolame 44 and E2 43. The most potent was EB (LQED50 of 4.1 +/- 0.5 microg/kg); then E2 10 microg +/- 2.2/kg; prolame 268 +/- 19 microg/kg; butolame 402 +/- 21 microg/kg, and pentolame 1037 +/- 28 microg/kg. The AEs LQ potency decreases as length substitution on the amine group in C-17 increases. AEs LQDE50 values correlate with previous Uw DE50, LH ID50 and binding studies indicates mediation of the response by estrogen receptors. AEs facilitate sexual behavior of Ovx rats as partial estrogenic agonists.

Morphometric analysis of mice uteri treated with the preservatives methyl, ethyl, propyl, and butylparaben.

The alkyl esters of p-hydroxybenzoic acid (PHBA) known as parabens (Pbens) are widely used as preservatives in food, pharmaceuticals, and cosmetics. Several in vivo and in vitro studies have shown these compounds to be estrogenic. Here, for the first time, we present evidence of their estrogenicity using a morphometric analysis of uteri from mice treated with the preservatives methylparaben (MePben), ethylparaben (EtPben), propylparaben (PrPben), and butylparaben (BuPben) compared with estradiol (E2). Different groups of adult ovariectomized (Ovx) CD1 mice were subcutaneously (sc) treated daily for three days with two different equimolar doses (362 and 1086 micromol/kg) of the Pbens: MePben (55 and 165 mg/kg), EtPben (60 and 180 mg/kg), PrPben (65 and 195 mg/kg), BuPben (70 and 210 mg/kg), E2 (10 microg/kg; 0.036 micromol/kg), and vehicle (propyleneglycol; V, 10 mL/kg). On the fourth day, uteri were dissected, blotted, weighed, and placed in a fixative solution for 24 h. The paraffin embeded uteri were cut to obtain 7 microm thick transversal sections. Luminal epithelium heights (LEH), glandular epithelium heights (GEH), and myometrium widths (MW) were measured. The highest Pbens dose was able to produce uterotrophic effects (38 to 76%) compared to E2 efects (100%). The relative uterotrophic potency to E2 (100) was from 0.02 to 0.009. Significant increases (P < 0.05) in LEH, GEH, and MW as compared with V were obtained: LEH from 87 to 113% (E2 153%), GEH from 10 to 40% (E2 60%), and MW from 35 to 43% (E2 88%). These results confirm that Pbens at the doses assayed here induce estrogenic histological changes in the uteri of Ovx mice.

In vivo estrogen bioactivities and in vitro estrogen receptor binding and transcriptional activities of anticoagulant synthetic 17beta-aminoestrogens.

Estrogenic activities of the two 17beta-aminoestrogen (AE) derivatives, prolame and butolame, were studied upon coagulation, serum luteinizing hormone (LH) and uterine weight, including endometrial morphology in castrated female rats. We have also investigated the ability of these two compounds, as well as another AE pentolame, to activate transcription through the estrogen receptor alpha (ERalpha) and the estrogen receptor beta (ERbeta). Administration of prolame and butolame to castrated animals increased significantly (P < 0.01) the mean clotting time when compared with that obtained in the group of control animals. Butolame was a more potent anticoagulant than prolame (P < 0.01), as judged by their corresponding IC(50) (5.4 +/- 0.65 and 66.6 +/- 2.57 micro;g/animal, respectively). In contrast, estradiol significantly shortened blood clotting times (P < 0.005). Both prolame and butolame caused a significant inhibition of serum LH levels (EC(50) 8.10 +/- 0.79 and 17 +/- 64 microg/animal, respectively), and restored castration-induced reduction in uterine weight of ovariectomized rats (EC(50) 4.14 +/- 1.57 and 17.0 +/- 1.78 microg/animal, respectively). In terms of the effects of prolame, butolame and pentolame in transient transfection assays, all the three AE activated ER dependent reporter gene expression, however, only at high concentrations. Prolame had the highest activity followed by butolame and pentolame. Induction of transcription by these compounds was preferentially mediated through the ERalpha, especially in the case of pentolame where little, if any, activation occurred through the ERbeta. None of the compounds showed antagonistic activities through either ER subtype. The overall data suggest that modifications in the structure and length of the amino-alcohol side-chain at C-17 might have an impact on the affinity and estrogenic intrinsic properties of AE at the level of diverse target tissues.

A comparative structural study of the steroid epimers: 17 beta-amino-1,3,5(10)-estratrien-3-ol, 17 alpha-amino-1,3,5(10)-estratrien-3-ol, and some derivatives by 1H NMR, and x-ray diffraction analysis.

The epimers 17 beta-amino-1,3,5(10)-estratrien-3-ol and 17 alpha-amino-1,3,5(10)-estratrien-3-ol were synthesized. 17 beta-Amino-1,3,5(10)-estratrien-3-ol was prepared by catalytic hydrogenation of the estrone-oxime. The 17 alpha-amino epimer was obtained from estradiol, via tosylate, followed by nucleophilic displacement by sodium azide and subsequent reduction with LiAlH4. They were characterized by spectroscopic methods. Determination of the crystal structures of 3-(toluene-4-sulfonyloxy)-17 alpha-1,3,5(10)-estratriene, 3-(toluene-4-sulfonyloxy)-17-alpha-amino,1,3,5(10)-estratriene hydrochloride, 17 beta-acetylamino-1,3,5(10)-estratriene-3-ol, and 3-acetoxy-17 beta-acetylamino-1,3,5(10)-estratriene enabled us to characterize the structure of the 17 alpha and 17 beta amino epimers for the first time.

Estrogenic effects of the synthetic aminoestrogen 17 beta-(5-hydroxy-1-pentylamino)-1,3,5(10)-estratrien-3-ol (pentolame).

In this study, we investigated the effects of pentolame, a 17 beta-aminoestrogen derivative, upon coagulation, serum LH, pituitary progestin receptors, uterine weight, and endometrium morphological changes in the castrated female rat. Groups of animals were subcutaneously (s.c.) injected with either estradiol (E2) (0.1 up to 1000 micrograms/animal), pentolame (1 up to 1000 micrograms/animal), or the vehicle alone daily for 5 consecutive days starting 2 weeks following ovariectomy. Administration of pentolame (10 to 1000 micrograms/animal) increased significantly (p < 0.05) the blood clotting time when compared with that obtained in the group of control animals (EC50 582 micrograms). Pentolame (500 and 1000 micrograms/rat for 5 days) caused a significant inhibition (p < 0.01) of serum LH levels (IC50 860 micrograms), which remained suppressed until Day 5 post last injection. In addition, treatment with pentolame was able to restore in the castrated female rat the presence of specific estrogen-dependent progestin binding sites at the anterior pituitary level. The affinity constants and the number of binding sites of pentolame-induced progestin receptors were similar to those obtained with estradiol at equipotent doses (860 micrograms vs. 1 microgram/animal, respectively). Administration of the 17 beta-aminoestrogen derivative resulted in a significant increase in uterine weight (EC50 420 micrograms) and endometrial characteristics were indistinguishable from those observed in the group of rats treated with E2.

Estrogenic effects of p-hydroxybenzoic acid in CD1 mice.

Xenobiotic estrogens in the environment or diet have received much attention as a possible source of certain hormonal disease states in human and wildlife. Therefore, the detection of estrogenic activity of any substance, especially those related to the food industry, is important. The estrogenic activity of p-hydroxybenzoic acid (PHBA), a compound related to a commonly used group of preservatives in food, cosmetic, and pharmaceutical preparations, was evaluated with immature and adult ovariectomized female mice (CD1) using two well-known bioassays. Subcutaneous administrations (s.c.) of different doses of PHBA were compared with estradiol (E2), and their effects on vaginal cornification and uterotrophic activities were evaluated. Different groups of animals were treated s.c. daily for 3 days with vehicle (corn oil, 0.3 ml/100 g), E2 (1 microgram/100 g), and PHBA (0.5, 5, 50, and 500 micrograms/100 g). Four days after treatment, PHBA produced a dose-dependent response on vaginal cornification and uterotrophic activity in both immature and adult ovariectomized mice. The relative uterotrophic potency of PHBA (500 micrograms/100 g) to E2 (1 microgram/100 g) was 0.0011 in immature mice and 0.0018 in ovariectomized animals.

Allopregnanolone potentiates a GABA-withdrawal syndrome in the rat cerebral cortex.

We have studied the neuromodulatory effect of the neurosteroid 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone-3 alpha-5 alpha P-) in the GABA-withdrawal syndrome (GWS). This is a model of partial epilepsy consisting of an enduring paraoxysmal activity recorded at the site of GABA infusion that depends, for its induction, on GABA receptor activation. Rats were chronically implanted for frontal and occipital EEG recording with infusion cannulae fixed on the somatomotor cortical region. When the neurosteroid was infused after or concurrently with GABA, a potentiation of the GWS (i.e. shorter latency and prolonged duration) was observed. No modifications in EEG activity were detected when allopregnanolone was administered alone or prior to GABA administration. These results indicate a neuromodulatory effect of allopregnanolone, dependent on the presence of GABA at the receptor site.

Anticoagulant and estrogenic effects of two new 17 beta-aminoestrogens, butolame [17 beta-(4-hydroxy-1-butylamino)-1,3,5(10)-estratrien-3-ol] and pentolame [17 beta-(5-hydroxy-1-pentylamino)-1,3,5(10)-estratrien-3-ol].

The syntheses and characterizations of two new 17 beta-aminoestrogens, butolame [17 beta-(4-hydroxy-1-butylamino)-1,3,5(10)-estratrien-3-ol] and pentolame [17 beta-(5-hydroxy-1-pentylamino)-1,3,5(10)-estratrien-3-ol], are presented. Both compounds, when administered in single subcutaneous injections to male mice and rats, produce dose-dependent increases in blood clotting times that may last several days. The estrogenic effects assessed by the vaginal cornification test are of relatively short duration.

The anticoagulant effect of hexolame, N-(3-hydroxy-1,3,5(10)-estratrien-17 beta-yl)-6-hydroxyhexylamine, another amino-estrogen with prolonged anticoagulant effect.

The anticoagulant and estrogenic effects of hexolame, N-(3-hydroxy-1,3,5(10)-estratrien-17 beta-yl)-6-hydroxyhexylamine, are described. A single subcutaneous injection of hexolame in adult and infant male mice produced dose-dependent increases in blood clotting time which could be observed even after 2 days. In ovariectomized mice, hexolame produced vaginal cornification (estrogenic response). The data suggested that if used in the treatment of prostatic cancer, hexolame, like prolame, would not induce cardiovascular accidents. It could also be useful in the prevention of thrombosis.

The anticoagulant effect of prolame, N-(3-hydroxy-1,3,5(10)estratrien-17 beta-yl)-3-hydroxypropylamine, a novel amino-estrogen.

The anticoagulant and estrogenic effects of prolame, N-(3-hydroxy-1,3,5(10)-estratrien-17 beta-yl)-3-hydroxypropylamine, are described. A single subcutaneous injection of prolame in male mice, ovariectomized mice, adult and infant male rats, produced dose-dependent increases of blood clotting time, which could be observed with the larger doses even after 4 days. In ovariectomized mice, prolame produced vaginal cornifications of shorter duration than those produced by estradiol-17 beta. The evidence suggests that, in contrast with currently used estrogens, prolame would not generate cardiovascular accidents if used for the treatment of prostatic carcinoma; it could also be exceptionally effective for the prevention of thrombosis.