Potential Estrogenic Properties of 17β-Hydroxy-ethylimine Estradiol Derivative Targeted to Menopause Stage

Abstract

Background/Aim: Hormone replacement therapy during menopause increases the risk of thromboembolic diseases and cancer, so safety alternative therapeutic strategies are needed. 17β-Aminoestrogens are a synthetic estrogens group that possess mild anticoagulant activity that contrasts with the pro-coagulant effects showed by estradiol’s (E₂) in rodents. Being considered an alternative to conventional hormone replacement therapy during menopause without thrombogenic risks producing. The present study aimed to determine the estrogenic profile and anxiolytic activity of 17β-[hydroxy-ethylimine]-1,3,5(10)-estratrien-3-ol (IE₂), a related compound unknown until now. Methods: IE₂ was assessed in immature rats by uterotrophic assay administering IE_2, E_2, or vehicle. In ovariectomized adult Wistar rats (Ovx) to facilitating the lordotic behavior compared with E_2, estradiol benzoate, or vehicle. The effect of IE₂ on anxiety was estimated in Ovx animals treated with IE₂, E₂, or vehicle group and evaluated in the elevated plus-maze model. Results and conclusion: IE₂ produced an uterotrophic effect, lordotic behavior, and anxiolytic effect in a dose-dependent manner, similar to E₂. IE₂ depicted estrogenicity, indicating potential clinical use as hormone replacement therapy during menopause.

INTRODUCTION

Sex steroid hormones are involved in the differentiation and maintenance of sexual characteristics and reproduction. The ovaries cyclically secrete estrogens from puberty to menopause; they profoundly influence women’s physiology and behavior. In perimenopause, estrogens deficiency produces physiological changes such as hot flashes, night sweats, irregular periods, insomnia, the decay of sexual motivation, also psychological changes as symptoms of anxiety, depression, and alterations in cognitive functions.¹⁾ Anxiety disorders have a population incidence of 5% that can increase up to 10% in women over 40 and are more severe during perimenopause and menopausal periods.²⁾ Estrogen levels reduction at this stage is an indicator of the participation of their modulation in the affective state. Hormone replacement therapy (HRT) with conjugated equine estrogens, ethinyl estradiol, and estradiol derivatives are used to manage peri- and menopause symptoms.³⁾ However, many clinical studies described the high risk of adverse effects associated with its use, such as thromboembolic events and cancer.⁴⁾ Thus, taking into account them, it is recommended to prescribe HRT at the minimum effective dose and for the shortest time possible under closed supervision.^5,6) Moreover, lifespan has increased in the human population, and the menopausal stage is a long period for most women to cope with the deficiency.

In the search for alternative synthetic estrogens without producing thromboembolic risk, our group has reported the biological characterization of the synthetic estrogens 17β-aminoestrogens. These compounds possess anticoagulant effects, which contrast with the pro-coagulant effects of 17β-estradiol (E₂) and other estrogen derivatives used in HRT. They behave as estrogenic partial agonists of E₂, increasing uterine weight, activating estrogen receptor transcription, and decreasing luteinizing hormone. Also, they induce progesterone receptors synthesis in the anterior pituitary gland and stimulate sexual behavior (lordosis) in the ovariectomized (Ovx) rat.⁷⁾ As part of the characterization of the activity profile of the 17β-aminoestrogens, the anxiolytic, anti-depressant, and mnemonic properties have been described in the Ovx rat.⁸⁾ The 17β-aminoestrogens low estrogenic properties in peripheral tissues, coupled to their enhanced activity in the central nervous system (CNS) without producing prothrombotic effects, could be an alternative HRT directed to menopausal women.

The objective of the present work was the evaluation of the estrogenic and anxiolytic effects of an imino estradiol derivative, 17β-[hydroxy-ethylimine]-1,3,5(10)-estratrien-3-ol (IE₂) (Fig. 1). A compound with a closed structural relation to 17β-aminoestrogens that has not been biologically evaluated until now.

Fig. 1. Chemical Structure of IE₂

MATERIALS AND METHODS

Reagents

17β-Estradiol (E₂; 1,3,5(10)-estratrien-3,17β-diol), 17β-estradiol benzoate (EB; 1,3,5(10)-estratrien-3,17β-diol 3-benzoate), and progesterone (P; Pregn-4-ene-3,20-diona) were purchased from Sigma Chemical (St. Louis, MO, U.S.A.). 17β-[Hydroxi, ethylimine]-1,3,5(10)-estratrien-3-ol (IE₂, Fig. 1), was prepared from estrone according to the Imscher method.⁹⁾ Chemical purity was established by chromatographic (HPLC, TLC) and spectral (IR/NMR/MS) techniques.

Animals

The study was carried out at the Laboratory of Endocrine Pharmacology, Department of Pharmacology, Faculty of Medicine, National Autonomous University of Mexico (UNAM), Mexico City, and approved by the Local Animal Ethics Committee. All experimental studies were conducted following the Mexican National Protection Laws of Animal Welfare (NOM-062-Z00-1999). Wistar immature female 21 d old (30–40 g), adult female, and male eight-nine weeks old (200–250 g) rats from our animal breeding facilities were used. In the beginning, groups of five rats were housed in standard local acrylic cages with autoclaved wood shaving bedding (90–100 g/cage). The immature female rats for the uterotrophic assay were kept in a room with a 12–12 h light–dark cycle (lights on from 8:00 to 20:00). Adult male and female Wistar rats evaluated for anxiety and facilitation of sexual behavior experiments were kept in a reversed 12–12 h light–dark cycle (lights off from 8:00 to 20:00). All animals were maintained with constant temperature (20–22 °C), humidity range of 50–10% fed with a commercial diet (Nutricubos, Purina), and tap water ad libitum. On completion of each experiment, rats were euthanized with an overdose of chloral hydrate anesthesia.

Evaluation of Uterotrophic Effect

Immature animals were distributed among groups according to a balanced design based on body weight. Groups (n = 8–10) were randomly assigned to treatment groups. Animals were subcutaneously (s.c.) injected once a day for 3 consecutive days with: E₂ (1, 10, 50, 100, 250, 500 µg/kg) or IE₂ (1.3, 6.7, 13, 55, 110 µg/kg), the control group received the vehicle (10 mL/kg) only. On the fourth day, the animals were weighed, and the uterotrophic activity induced by the tested compounds was evaluated by the gain in uterine wet (Uww)¹⁰⁾ and dry weight (Udw). The uteri were dissected, blotted, and weighed to obtain the Uww. Afterward, the organs were dried at 36 ᵒC for 24 h and weighed again to obtain the Udw. The uterine weights of all the groups were expressed in milligrams. The percent of response was calculated with relation to the vehicle (100%).

Ovariectomy

Three weeks before hormonal treatment, adult female Wistar rats were bilaterally ovariectomzed, under chloral hydrate anesthesia (4% solution, 280 mg/kg; J.T. Baker, Mexico City, Mexico). After the ovariectomy recovery period, the animals were randomly assigned to the different groups.

IE₂ vs. E₂ an EB Treatment and Lordosis Quotient (LQ) Behavioral Test Design

All steroids (E₂, EB, and IE₂) were dissolved in corn oil as the vehicle (V) and s.c. injected in a total volume of 300 µL/rat (approx. 1.2 mL/kg/d). Drug administrations were at 8:00 h, (reversed cycle dark phase). Dose–response curves of LQ of E₂, EB, and IE were performed (n = 8–10 animals per group). The dose–response curves were obtained with different groups of Ovx rats. Single injections of different doses of E₂ (0.3, 3, 30, 60, 300 µg/kg), or EB (0.4, 4, 40, 80, 400 µg/kg), or the IE₂ (27, 55, 110, 220, 440 µg/kg), the control (V) group received 300 µL/rat/d (approx. 1.2 mL/kg/d). After 24 h, P (1 mg/rat dissolved in 100 µL of the V (4 to 5 mg/kg) was injected. Sexual receptivity was tested at hour 0 before the first administration, then, 24 h after it (before P administration), and after 29 h (5–7 h after P administration).

LQ Behavioral Evaluation

All the experiments were done under dim red light and preformed, as previously described.¹¹⁾ Sexual receptivity was evaluated from each female, which was placed in a testing arena (cylindrical transparent plastic cage, diameter = 40 cm, height = 40 cm) with a sexually experienced male (males were acclimated into the arena for 3 min before testing). The males were allowed to mount the females ten times or for 10 min. The number of lordotic female responses (head lifting, arching the back, and tail movement of one side) to male mounting was recorded by a researcher blind to the experimental treatment. For each female, the LQ (number of lordosis displayed/number of mounts ×100) was calculated as a measure of sexual receptivity.

Elevated Plus-Maze Experimental Design (EPM)

Four groups of Ovx rats (n = 8–10 animals/group) were treated by s.c. administration for 3 consecutive days of E₂ (25 µg/kg/d), IE₂ (55 or 110 µg/kg/d) and the V group (approx. 1.2 mL/kg/d). On the fourth day, the assessment was made between 8.00 and 12.00 h by one or two experienced testers who were blind using the EPM described previously.¹²⁾ The EPM model is based on the rodents’ natural fear of open spaces. The model does not involve nociceptive or conditioning procedures. The EPM employed consisted of four connected black Plexiglass arms each 10 cm wide and 50 cm long and elevated 50 cm off the ground. Two arms were enclosed by walls 30 cm high, and the other two arms were open.

Rats were placed in the central area (at the intersection of the four arms) and were video recorded for 5 min. The capture and processing of the behavioral parameters were obtained through the OAVid-Red 2013 Software. The time the rats spent in the open and closed arms was measured. The number of entries in the closed or open arms of the maze was recorded when the rat had all four paws on the closed or open arms of the EPM only. At the end of each rat evaluation, the maze was cleaned with ethyl alcohol (60%) to avoid odor interference.

Data Analysis

The results were expressed in means ± standard error of the mean (S.E.M.). Percent differences with respect to the vehicle group were obtained by the relation: [Treated group (100)/control group]. The relative effects of E₂ were calculated by the relation: [E_max IE₂]100/(E_max E₂). The dose–response curves for E₂, BE and IE₂ were fitted to a sigmoid curve, calculated by the Hill equation: Y = min + (max-min) [ Xⁿ/Kⁿ + Xn, where min is the baseline response, max correspond to the maximal response, X represents the log dose, K is ED₅₀ and n the Hill coefficient. The ED₅₀ and the maximum response value (E_max) were obtained by the software Origin 8.0 (OriginLab Corporation, Northampton, MA, U.S.A.). Statistical significance among the different treated groups was estimated by a one-way ANOVA test. The significance of the differences between control (vehicle) and treated groups was assessed by the Post Hoc test (Tukey, Dunn’s, or Dunnet’s methods) as required. p < 0.05 was considered as the limit for statistical significance. The analysis was performed using the Sigma Plot software 11.0 (Jandel Corporation).

RESULTS

IE₂ Uterotrophic Effect

IE₂ produced uterotrophic effects in immature rats. Figure 2 shows a significant dose-dependent increase (p < 0.05 vs. vehicle) in the uterine weight of prepubertal rats treated with different doses of IE₂ and E₂. The maximum response and the ED₅₀ of each compound were obtained from the dose-response curves and are described in Table 1.

Fig. 2. Dose–Response Curves of the Effect of IE₂ on the Uterine Wet Weight (Uww, Plot A) and Uterine Dry Weight (Udw, Plot B) of Immature Rats

As controls, estradiol (E₂), and the vehicle, corn oil were used (starting point). Each point represents the mean ± standard error of 8–10 animals. IE₂ showed a higher response in both Uww and Udw with respect to the E₂ (IE₂ E_max 138 mg ±17 vs. E₂ E_max 103 mg ±5), (IE₂ E_max 42 mg ±6 vs. E₂ E_max 20 ± 1). In contrast, IE₂ DE₅₀ was higher in both Uww, (11.6 ± 1.2 µg/kg) and Udw, (12.3 ± 1.5 µg/kg) related to E₂ (9.6 ± 2.3 and 9.4 ± 2 µg/kg, respectively), which demonstrated lower potency than E₂.

Table 1. Uterotropic Effect ED₅₀ and Emax of E₂ and IE₂ of Prepubertal Wistar Rats

	Uww			Udw
	Emax (mg ± S.E.M.)	DE50 (µg/kg)	Relative effect (%)	Emax (mg ± S.E.M.)	DE50 (µg/kg)	Relative effect (%)
Vehicle	22 ± 0.9	—	—	4 ± 0.8	—	—
E2	103 ± 5	9.6 ± 2.3	100	20 ± 1	9.4 ± 2.0	100
IE2	138 ± 17	11.6 ± 1.2	121	42 ± 6	12.3 ± 1.5	131

Uww = uterine wet weight; Udw = uterine dry weight. E_max: Maximum effect of uterine weight. DE₅₀: the dose of E₂ or IE₂ to increase 50% of the uterine weight. Relative effect to E₂ = [E_max IE₂]100/(E_max E₂).

IE₂ LQ Determination

The data about the effect of the single administration of E₂, EB, IE_2, and the control group on the sexual receptivity of Wistar Ovx rats are depicted in Fig. 3 and Table 2. The administered steroids and subsequent sequential administration of P facilitated the lordotic response significantly (p < 0.05 vs. V). The maximum response and LQED₅₀ of the reference E₂, EB, and IE₂ were obtained from their corresponding dose-response curves (Fig. 3, Table 2). EB had the highest efficacy as a facilitator of sexual receptivity in the Ovx rat with a maximum lordosis quotient (LQE_max) response of 92%, close to that previously described,¹¹⁾ followed by IE₂ with an 86% and E₂, which had the lowest efficacy of 53%. The LQED₅₀ of EB was 10.9 µg/kg, showing to be more potent than E₂ and IE₂ (20.9 and 128.8 µg/kg, respectively). The results obtained by this model indicate that IE₂ exhibits an estrogenic activity with higher efficacy than E₂ to induce sexual receptivity in Ovx rats; however, with lower potency.

Fig. 3. Dose–Response Curves of the Effect of IE₂ in the Sexual Behavior of Female Rats

Different groups of Ovx rats were treated with a single administration of different doses of IE₂, as controls estradiol benzoate (EB) estradiol (E₂) and the vehicle included as a starting point in the graph. Their efficacy order was EB > IE₂ > E₂. Meanwhile their potency order was: EB > E₂ > IE₂. Each point represents the mean ± standard error of 8–10 animals per group.

Table 2. LQ E_max, LQDE₅₀ of E_2, EB, IE₂ in the Ovx Rats

Group	LQ Emax (LQ + S.E.M.)	LQ ED50 (µg/kg)	Relative effect
Vehicle	0	0
E2	53 ± 10	20.9 ± 1.3	100
EB	92 ± 11	10.9 ± 1.2	180
IE2	86 ± 12	128.8 ± 6.0	165

LQE_max = mean maximum LQ ± S.E.M.; LQED₅₀ = effective dose to produce LQ₅₀. Relative effect to E₂ = [E_max EB or IE₂]100/(E_max E₂).

IE₂ Evaluation in the EPM

The EPM test was carried out to assess the anxiolytic effect of IE₂ compared to that produced by E₂ in Ovx rats. The groups treated with E₂ (25 µg/kg), IE₂ (55 and 110 µg/kg) maintained a time spent into open arms 38, 43 and 45% respectively, which were significantly longer than those observed in the control group (V = 24%; Fig. 4A). Consequently, the time spent by the Ovx rats different groups into closed arms was as follows: E₂ 30%, IE₂ (55) 35% and IE₂ (110 µg/kg) 28%, contrasting with the control group that remained 46% of the time in closed arms (Fig. 4B). The ANOVA analysis showed significant differences between the groups (F_(3.31) = 5.79, p < 0.003). The Post Hoc Tukey test revealed that the E₂ group and the two IE₂ groups (55 and 110 µg) spent a long time significantly into the open arms than the control group (Fig. 4A). Consequently, E₂ and IE₂ groups spent less time in the closed arms related to the control group significantly (ANOVA, F_(3.31) = 6.05, p < 0.002) (Fig. 4B). The difference between the total number of entries into the open and the closed arms by the groups did not show significant differences (Figs. 4C, D).

Fig. 4. The Anxiolytic Effect of IE₂ in the EPM

The values represent the mean ± standard error of the percentages of time that the different groups of animals spent into open arms (a), or closed arms (B) and the entries number into the open arms (C) and closed arms (D) for 5 min. Each point represents the mean ± standard error of 8–10 animals per group. * p < 0.05 vs. control.

DISCUSSION

The present study shows that IE₂ produced high estrogenic activity in Ovx rats using two classical paradigms for estrogenic activity evaluation: the uterotrophic assay, as well as an inducer of the sexual behavior of the rat. IE₂ in the uterus of immature mice induced a dose-dependent increase in uterine weight similar to that exerted by E₂, a phenomenon that is consistent with other reports.¹³⁾ Its uterotrophic potency evaluated by the wet uterine weight (Uww) was higher than that exhibited by E₂. Probably IE₂, in the same way as E₂, will also increase the uterine tissue (endometrium, myometrium, luminal epithelium, etc.), in all likelihood acting through the activation of estrogen receptors (ERs) by stimulating the transcription of specific genes in reproductive function. The higher uterotrophic potency of IE₂ contrasts with the effects observed in the Ovx rats treated with 17β-aminoestrogens, which have a very low uterotrophic efficacy and potency.¹⁴⁾ These compounds are modified estrogens where the C17 has a -N-[CH₂]n-OH group, causing the estrogenic activity to decrease as the number of methylenes increases. The structural change significantly impacts their estrogenic potency, being up to 500 times lower than that of E₂ when n = 5 in uterotrophic trials in young Ovx rats and immature rats and mice.¹⁴⁾ The low estrogenic activity is related to the lower affinity of 17β-aminoestrogens for the estrogen intracellular receptors ERα and ERβ concerning E₂.¹⁵⁾ The chemical structure of IE₂ contains the group R = N-[CH₂]₂-OH, where R = C17 of the steroidal ring similar to that of E₂, which favoring uterotrophic efficacy and potency and could be indicating a higher affinity for IE₂ intracellular ERs related to that exhibited by the 17β-aminoestrogens.

On the other hand, IE₂ produces effects similar to those induced by E₂ at the CNS level. The administration of E₂ and IE₂ sequentially with P induced a lordotic response in young Ovx rats in a dose-dependent manner. The maximum response of IE₂ (LQ = 86%) was more effective than E₂ (LQ = 53%) although, its relative potency was lower (ED₅₀; IE₂ 129 and E₂ 21 µg/kg). These results show that IE₂ acts on sexual receptivity in young Ovx rats in a similar manner to that induced by E₂. Treatment with E₂ and P modulates the neural activity and induces proceptive and receptive behaviors in Ovx rats.¹⁶⁾ However, the control of lordotic reflex in the rat is a complex issue. Several systems and events are coordinated at the hypothalamic level in the ventromedial nucleus (VMH), medial preoptic nucleus (MPN), and mesencephalic level periaqueductal gray (PAG) matter. Which modulate and integrate hormonal, neurochemical, and sensory behavioral information.¹⁷⁾ Many neurotransmitter systems (serotonin, dopamine, norepinephrine, γ-aminobutyric acid (GABA), etc.) and neuropeptides located in the brain areas are involved in the control of sexual receptivity.¹⁸⁾ The receptive sexual behavior (lordosis), induced by E₂, in the rat is related to the increased progesterone receptors synthesis by genomic events due to the gene transcription stimulation, which increases the P effectiveness. However, non-genomic effects occur at the membrane level, such as the ion flow and neurosteroid action. The 5α-pregnan -3α-ol-20-one (3α, 5α-THP; also known as allopregnanolone) in the ventral tegmental area (VTA); is de novo synthesized from cholesterol in both the CNS and the peripheral system from its precursor P, which concentration is independent of the endocrine gland secretion.¹⁹⁾ Neurosteroids act in the brain through ionotropic receptors, being positive allosteric modulators of GABA_A receptors.²⁰⁾ In VTA, 3α, 5α-THP activates the GABA_A receptor and prolongs the Cl⁻ channels opening, which leads to an increase in the duration of inhibitory postsynaptic potential.²¹⁾

The GABAergic system, as the primary inhibitor of neuronal excitability of vertebrates, is involved in the regulation of fear and anxiety. The anxiolytic effects of E₂ in female ovariectomized rodents have been demonstrated. Ovariectomy decreased E₂ and P levels. In the brain of Ovx animals, GABA_A levels, along with the number of GABAergic receptors, is decreased. The Ovx animals display anxiety, and depressive-like behaviors, which can be reverted by the sequential administration of E₂ and P, or allopregnanolone.^22,23)

IE₂ produced anxiolytic effects similar to those observed in Ovx rats treated with E₂ obtained through the EPM paradigm.²⁴⁾ Animals treated with IE₂ spent more time in the open arms of the EPM maze related to the control group, and its effects were very similar to those observed in animals treated with E₂. Under normal conditions, rodents enter the closed arms and avoid the open arms of the EPM. Anxiolytics agents such as diazepam, increase the rodent’s permanence time in open arms, which is considered an anxiolytic-like effect.²⁵⁾ Some hormonal agents such as P, 5α-THP, and estrogens like E₂ are anxiolytic in the EPM.²⁶⁾

On the other hand, also is the consideration estrogens influence on the mono-aminergic neurotransmitter systems (serotonin, norepinephrine, and dopamine) involved in the pathophysiology of affective disorders, such as depression.²⁷⁾ Since E₂ modulates biosynthesis, transport, and metabolism, determining the number of receptors in their activity area.^28,29) The IE₂ anti-anxiety properties mechanism needs to be studied to increase the evidence on its estradiol-like anti-anxiety effects.

CONCLUSION

The results showed here, particularly the IE₂ induction of lordotic behavior and its anti-anxiety properties show its capability to reach CNS exerting properties similar to that shown by E₂. Our results open the way for further research on the effects of IE₂ or steroidal imines substituted at the C17 as anxiolytic agents. Furthermore, it is no prothrombic effects, cardiovascular effects, or cellular promotion of hormone-dependent malignancies would also have to be considered for future studies. They encourage abord toxicological studies, biological targets, and mechanisms of action at the CNS level to know their potential clinical use associated with anxiety drugs or depression disorders during the menopause period.

Acknowledgment

The authors are grateful to Dr. Lorena Mendiola Almaraz for the animals’ care during the experiments and her technical assistance.

Conflict of Interest

The authors declare no conflict of interest.

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