Pharmacology of estradiol

Mechanism of action

Estradiol is an estrogen, or an agonist of the nuclear estrogen receptors (ERs), the estrogen receptor alpha (ERα) and the estrogen receptor beta (ERβ).^[7][8][12] According to one study, the EC₅₀ value of estradiol for the human ERα is 50 pM (0.05 nM) and for the human ERβ is 200 pM (0.2 nM).^[8][13] Estradiol is also an agonist of the membrane estrogen receptors (mERs), including the G protein-coupled estrogen receptor (GPER), G_q-coupled membrane estrogen receptor (G_q-mER), ER-X, and ERx.^[14][15] It is far more potent as an estrogen than are other natural and bioidentical estrogens like estrone and estriol.^[7]

Estradiol has little to no affinity for other steroid hormone receptors, including the androgen, progesterone, glucocorticoid, and mineralocorticoid receptors.^[16][17][18] It has weak affinity for the androgen receptor, with about 8% of relative binding affinity of testosterone according to one study,^[19] and shows agonistic activity at this receptor.^[20] However, estrogens circulate in the picomolar range while androgens circulate in the nanomolar to micromolar range,^[21][22] and in accordance with this, estradiol is active as an estrogen in target tissues at approximately 1,000-fold lower concentrations than is testosterone.^[23] In addition, while estradiol did show activation of the androgen receptor in vitro at very high concentrations, its efficacy as an androgen receptor agonist was of such low potency that it was not possible to calculate an EC₅₀ value for the activity.^[20] As such, the weak activity of estradiol at the androgen receptor is unlikely to be of biological significance at normal physiological concentrations.^[19][20]

The affinities of estradiol for the ERs are high (around 0.1 nM), and there is a relatively low quantity of about 10,000 to 20,000 ERs in the cytoplasm per cell in estrogen target tissues.^[24] Estradiol stays bound to the ERs for about 24 hours, which is longer than that of other estrogens such as estriol (6 hours).^[7] A prolonged duration of binding to the ERs (e.g., 9 to 12 hours for endometrial effects), as with estradiol, is necessary for full estrogenic responses in various tissues.^[7] The ERs downregulate with exposure to estradiol, and in accordance, the expression of the ERs is dependent on estradiol concentrations.^[25][26] Constant levels of estradiol may result in downregulation of the ERs and relatively diminished responses to estradiol, although this has not been assessed clinically.^[25] Once bound to estradiol, the ERs are ubiquitinated and degraded by proteasomes, which is involved in ER downregulation.^[26] The unbound ERα has an intracellular half-life of up to 5 days, but this shortens to 3 to 4 hours once bound to a ligand such as estradiol.^[27][26] Estrogen deprivation can easily increase sensitivity to estrogens like estradiol by 10,000-fold or more, demonstrating a profound capacity of the ERs for upregulation and downregulation.^[28] This increase in sensitivity is mediated by a 100-fold increase in ERs, as well as other mechanisms such as changes in coactivator sensitivity and degree of phosphorylation of transactivation factors.^[28] Progestogens like progesterone and androgens like testosterone downregulate the ERs in certain tissues such as the endometrium and breasts, among others.^[29][7][30]

Effects in the body and brain

The ERs are expressed widely throughout the body, including in the breasts, uterus, vagina, prostate gland, fat, skin, bone, liver, pituitary gland, hypothalamus, and elsewhere throughout the brain.^[33] Through activation of the ERs (as well as the mERs), estradiol has many effects, including the following:

• Promotes growth, function, and maintenance of the breasts, uterus, and vagina during puberty and thereafter^[33][34]
• Mediates deposition of subcutaneous fat in a feminine pattern, especially in the breasts, hips, buttocks, and thighs^[35]
• Maintains skin health, integrity, appearance, and hydration and slows the rate of aging of the skin^[36]
• Produces the growth spurt and epiphyseal closure in both sexes during puberty, mediates widening of the hips in females during puberty, and maintains bone mineral density in both sexes throughout life^[37][38]
• Modulates hepatic protein synthesis, such as the production of sex hormone-binding globulin (SHBG) and numerous other proteins, with consequent effects on the cardiovascular system and various other systems^[9]
• Exerts negative feedback on the hypothalamic–pituitary–gonadal axis (HPG axis) by suppressing the secretion of the gonadotropins FSH and LH from the pituitary gland, thereby inhibiting gonadal sex hormone production as well as ovulation and fertility^[39][9][40]
• Regulates the vasomotor system and body temperature via the hypothalamus, thereby preventing hot flashes^[41][42]
• Modulates brain function, with effects on mood, emotionality, and sexuality, as well as cognition and memory^[43]
• Influences the risk and/or progression of hormone-sensitive cancers including breast cancer, prostate cancer, and endometrial cancer^[44][9]

Estrogen has also been found to increase the secretion of oxytocin and to increase the expression of its receptor, the oxytocin receptor, in the brain.^[22] In women, a single dose of estradiol has been found to be sufficient to increase circulating oxytocin concentrations.^[45]

Antigonadotropic effects

Estradiol and testosterone levels over the course of 12 weeks following a single intramuscular injection of 320 mg polyestradiol phosphate in men with prostate cancer.^[46]

Estradiol and testosterone levels with high-dosage transdermal estradiol in the form of two to six 100 µg/day estradiol patches (Progynova TS forte) in men with prostate cancer.^[11][47][48]

Testosterone levels over the course of 6 months with intramuscular injection of different doses of polyestradiol phosphate once every 4 weeks in men with prostate cancer.^[10]

Testosterone levels with intramuscular 100 mg/month estradiol undecylate or 300 mg/week cyproterone acetate in men.^[49] Solid lines are the average levels and dashed lines are the highest and lowest observed levels.^[49]

Testosterone levels as a function of estradiol levels during 1 to 8 mg/day oral estradiol therapy in transgender women.^[50]

Estrogens are powerful antigonadotropins at sufficiently high concentrations.^{[40][51][52][10][11]} By exerting negative feedback on the hypothalamic–pituitary–gonadal axis (HPG axis), they are able to suppress the secretion of the gonadotropins, LH and FSH, and thereby inhibit gonadal sex hormone production and circulating sex hormone levels as well as fertility (ovulation in women and spermatogenesis in men).^[40][51][52] Clinical studies have found that in men treated with them, estrogens can maximally suppress testosterone levels by about 95% or well into the castrate/female range (< 50 ng/dL).^[10][11] This is equivalent to the reduction in testosterone levels achieved by orchiectomy and gonadotropin-releasing hormone analogue (GnRH analogue) therapy, corresponding to a complete shutdown of gonadal testosterone production.^[53][54] In addition, it is greater than that achieved with high-dose progestogens like cyproterone acetate and gestonorone caproate, which can maximally suppress testosterone levels in men by about 75%.^{[55][56][49][57][58]}

Suppression of testosterone levels by estradiol to within the castrate/female range (< 50 ng/dL) in men requires relatively high levels of estradiol and has been associated with circulating levels of 200 to 300 pg/mL and above.^[10][11] However, although the castrate range in men has been defined as testosterone concentrations of less than 50 ng/dL, mean levels of testosterone with surgical castration are actually about 15 ng/dL.^[59] To achieve such levels of testosterone with estradiol therapy, higher concentrations of estradiol of about 500 pg/mL have been necessary to produce the requisite maximal suppression of testosterone production.^[10] Injected estradiol esters like polyestradiol phosphate, estradiol valerate, and estradiol undecylate, as well as high-dose estradiol transdermal patches, are used as a form of high-dose estrogen therapy to suppress testosterone levels into the castrate range in men with prostate cancer.^{[9][60][61][62][11][49]} High dosages of estradiol in various forms and routes are also been used to suppress testosterone levels in transgender women.^[63][64][65]

Lower dosages and concentrations of estradiol can also significantly suppress gonadotropin secretion and testosterone levels in men and transgender women.^[66][67] A retrospective study of oral estradiol monotherapy in transgender women found that dosages of 1 to 8 mg/day increased mean estradiol levels to about 50 to 150 pg/mL and suppressed mean testosterone levels to about 120 to 10 ng/dL.^[50] However, there was high interindividual variability in the estradiol and testosterone levels achieved, and testosterone levels were insufficiently suppressed in many even at 8 mg/day.^[50] In another study, a dosage of 1 mg/day oral micronized estradiol in healthy older men, which increased circulating estradiol levels by a relatively high amount of 6-fold (to 159 pg/mL), estrone levels by 15-fold (to 386 pg/mL), and SHBG levels by 17%, was found to suppress total testosterone levels by 27% (to 436 ng/dL) and free testosterone levels by 34% (to 11.8 ng/dL).^[66][67]

Generally, estrogens are antigonadotropic and inhibit gonadotropin secretion.^[68][69] However, in women, a sharp increase in estradiol levels to about 200 to 500 pg/mL occurs at the end of the follicular phase (mid-cycle) during the normal menstrual cycle and paradoxically triggers a surge in LH and FSH secretion.^[68][70][69] During the mid-cycle surge, LH levels increase by 3- to 12-fold and FSH levels increase by 2- to 4-fold.^[71][72][73] The surge lasts about 24 to 36 hours and triggers ovulation, the rupture of the dominant ovarian follicle and the release of the egg from the ovary into the oviduct.^[68] This estrogen-mediated gonadotropin surge effect has also been found to occur with exogenous estrogen, including in post-hormone therapy transgender women and pre-hormone therapy transgender men acutely challenged with a high dose of an estrogen, but does not occur in men, pre-hormone therapy transgender women, or post-hormone therapy transgender men, hence indicating a hormonally-based sex difference.^[74] A sufficient amount of progesterone (corresponding to levels greater than 2 ng/mL) or a progestin prevents the mid-cycle estradiol-induced surge in gonadotropin levels in women.^[75][76] This is how progestins prevent ovulation and primarily mediate their contraceptive effects in women.^[76]

Effects on adrenal androgen levels

In addition to their antigonadotropic effects, estrogens at high concentrations can significantly decrease androgen production by the adrenal glands.^[9][77][78] A study found that treatment with a high dosage of 100 µg/day ethinylestradiol reduced circulating adrenal androgen levels by 27 to 48% in transgender women.^[9][77][78] Another study found similar effects in men with prostate cancer, with levels of the adrenal androgens dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), and androstenedione (A4) all decreasing significantly more with high-dose estrogen therapy (oral ethinylestradiol plus intramuscular polyestradiol phosphate) than with orchiectomy (by 33–39% and 10–26%, respectively).^[79]

However, a study found that these effects occurred with high-dose potent oral and synthetic estrogens such as ethinylestradiol and estramustine phosphate but not with the parenteral estrogen polyestradiol phosphate, suggesting that decreases in adrenal androgen levels are secondary to changes in liver protein synthesis rather than due to a direct action in the adrenal cortex, and that such changes will only occur in the context of strong hepatic impact.^[80][81] Cortisol levels were unchanged in the other groups (e.g., orchiectomy, GnRH agonist therapy, and parenteral estrogen therapy) in this study, but increased by 300 to 400% in the oral and synthetic estrogen groups, likely secondary to increases in hepatic corticosteroid-binding globulin (CBG) production and compensatory upregulation of adrenal corticosteroid synthesis.^[81]

Effects on liver protein synthesis

Estradiol and other estrogens modulate liver protein synthesis via activation of hepatic ERs.^[7] Estradiol increases the production and by extension circulating levels of sex hormone-binding globulin, corticosteroid-binding globulin (SHBG), angiotensinogen (AGT), pregnancy zone protein (PZP), coagulation factors, and numerous other hepatic proteins.^[7] Conversely, estradiol decreases hepatic synthesis and by extension circulating levels of insulin-like growth factor 1 (IGF-1).^[7] The effects of estradiol on liver protein synthesis are moderated by route of administration, with oral administration having 4- to 5-fold stronger effects on liver protein synthesis than doses by the transdermal route with equivalent general/systemic estrogenic potency.^[7] The influences of estradiol on liver protein synthesis have a variety of effects in the body, with implications for the bioavailability of androgens and the cardiovascular system.^[7]

Differences from other estrogens

Changes in levels of estrogen-sensitive proteins after treatment with oral estradiol or oral ethinylestradiol in postmenopausal women.^[54][82] FSH is a pituitary protein and represents general/systemic estrogenic effect, while SHBG and PZP are hepatic proteins and represent liver estrogenic effect.^[54][82]

SHBG levels in men with 1) intramuscular injection of 320 mg polyestradiol phosphate once every 4 weeks alone; 2) the combination of intra- muscular injection of 80 mg polyestradiol phosphate once every 4 weeks plus 150 µg/day oral ethinylestradiol; 3) orchiectomy only.^[10]

Estradiol has relatively low oral bioavailability of about 5%.^[7] In addition, there is considerable interindividual variability in levels of estradiol achieved with oral estradiol.^[7] In contrast to estradiol, the synthetic estrogen ethinylestradiol has about 45% oral bioavailability, around 80- to 200-fold greater systemic oral estrogenic potency, roughly 500- to 1,500-fold greater hepatic oral estrogenic potency, and less interindividual variability in circulating estrogen levels achieved.^{[54][7][1][83][84][85][86]} An oral dose of ethinylestradiol that is approximately 100-fold lower than that of estradiol achieves similar maximal circulating estrogen concentrations (e.g., 50 pg/mL ethinylestradiol with a single 20 μg dose of ethinylestradiol relative to 40 pg/mL estradiol with a single 2 mg dose of micronized estradiol or estradiol valerate).^[7] These differences are due to the introduction of an ethynyl group at the C17α position in ethinylestradiol (also known as 17α-ethynylestradiol), which results in steric hindrance and greatly diminishes the first-pass metabolism of ethinylestradiol relative to estradiol with oral administration.^[7] Estradiol and ethinylestradiol have similar affinities for and efficacies as agonists of the ERs,^[7][8] and the systemic estrogenic potency of estradiol and ethinylestradiol is similar when they are administered by the intravenous route.^[3]

Synthetic estrogens like ethinylestradiol and diethylstilbestrol and the natural but animal-derived conjugated estrogens have disproportionate effects on liver protein synthesis relative to their effects in other tissues when compared to estradiol.^[7] At doses via the oral route with comparable systemic estrogenic potency, conjugated estrogens have about 1.3 to 4.5 times the hepatotropic potency (i.e., potency in modulating liver protein synthesis) of estradiol, ethinylestradiol has about 2.9 to 5.0 times the hepatotropic potency of estradiol, and diethylstilbestrol shows about 5.7 to 7.5 times the hepatotropic potency of estradiol (all measured via a small selection of estrogen-modulated hepatic proteins that included HDL cholesterol, SHBG, CBG, and angiotensinogen).^[7] The greater hepatotropic potency of these estrogens relative to estradiol is related to susceptibility to hepatic metabolism.^[7] Whereas estradiol is metabolized and thereby inactivated rapidly upon entry into the liver, other estrogens like ethinylestradiol and diethylstilbestrol are resistant to hepatic metabolism and persist in the liver for a longer amount of time.^[7] This is reflected in the biological half-lives of these estrogens; the blood half-life of estradiol is about 1 to 2 hours, while the half-lives of ethinylestradiol and diethylstilbestrol are approximately 20 hours and 24 hours, respectively.^[2][87][86] In accordance with its long half-life, ethinylestradiol passes through the liver many times prior to its elimination.^[88] Because humans are not adapted to efficiently metabolize conjugated estrogens (which are equine (horse) estrogens) and synthetic estrogens like ethinylestradiol and diethylstilbestrol, these estrogens are not properly inactivated in the liver, with markedly disproportionate hepatic estrogenic effects resulting.^[7]

In addition to differences in hepatotropic potency between estradiol and other estrogens, there are differences in hepatotropic potency between different routes of administration of estradiol.^[7] Due to the first pass through the liver, oral estradiol results in disproportionate and unphysiological hepatic estradiol levels that are 4- to 5-fold higher than in the circulation.^[89][7] Conversely, parenteral routes of estradiol, such as transdermal, vaginal, and injection, bypass the first pass through the liver and produce levels of estradiol in the circulation and liver that are comparable.^[89][7] As an example of the reduced hepatic impact of parenteral estradiol relative to oral estradiol, a study found that 1 mg/day oral estradiol significantly increased SHBG levels by 45%, while 50 µg/day transdermal estradiol increased SHBG levels non-significantly by only 12% (with these dosages being roughly equivalent in systemic estrogenic potency).^[90][91][92] As such, not only do oral non-bioidentical estrogens like ethinylestradiol and conjugated estrogens have substantially greater potency in the liver than does oral estradiol, oral estradiol has considerably greater potency in the liver than does parenteral estradiol.^[7] Thus, the hepatotropic effects of oral non-bioidentical estrogens like ethinylestradiol are massive in comparison to parenteral estradiol (see the graph above/to the right), which in contrast to these estrogens has very weak or even absent effects on liver protein synthesis at normal therapeutic dosages.^{[7][46][54][9]} Whereas high-dosage 320 mg/month intramuscular polyestradiol phosphate increased SHBG levels to 166% in men with prostate cancer, the combination of 80 mg/month intramuscular polyestradiol phosphate and high-dosage 150 µg/day oral ethinylestradiol increased levels of SHBG to 617%, an almost 8-fold difference in increase and almost 4-fold difference in absolute levels between the two treatment regimens.^[10][46][93]

The effects of estrogens on liver protein synthesis, such as on the synthesis of coagulation factors, lipoproteins, and triglycerides, can cause an increased risk of thromboembolic and cardiovascular complications, which in turn can result in increased mortality.^[54] The risk of thromboembolic and cardiovascular complications is significantly increased in postmenopausal women taking oral conjugated estrogens as a component of menopausal hormone therapy.^[7][94][95] Both oral estradiol and oral esterified estrogens have been found to have a significantly lower risk of thromboembolic and cardiovascular complications than oral conjugated estrogens, and transdermal estradiol appears to have no such risks at all.^{[7][96][94][95]} Widely employed in the past, oral synthetic estrogens like ethinylestradiol and diethylstilbestrol are no longer used in menopausal hormone therapy due to their high risks of thromboembolic and cardiovascular complications.^[97] Studies have found a markedly increased 5-year risk of cardiovascular mortality of 14 to 26% in men treated with high-dosage oral synthetic estrogens like ethinylestradiol and diethylstilbestrol for prostate cancer.^[54] With diethylstilbestrol, there is an up to 35% incidence of cardiovascular toxicity and an up to 15% incidence of venous thromboembolism.^[98] In a small study comparing high-dosage 320 mg/month intramuscular polyestradiol phosphate versus the combination of 80 mg/month polyestradiol phosphate with high-dosage 150 µg/day oral ethinylestradiol for prostate cancer, there was a 25% incidence of cardiovascular complications over the course of a year in the group that was also treated with ethinylestradiol, whereas there were no cardiovascular complications in the polyestradiol phosphate-only group.^[46] In accordance, another study found no change in levels of coagulation factor VII, a protein of particular importance in the cardiovascular side effects of estrogens, with 240 mg/month intramuscular polyestradiol phosphate.^[99] In spite of the markedly reduced impact of parenteral estradiol on the liver compared to other estrogens however, high dosages of parenteral estradiol, producing high levels of circulating estradiol, can still result in important and undesirable changes in liver protein synthesis as with other estrogens.^[40] A high dosage of 320 mg/month polyestradiol phosphate has been found to result in significantly increased cardiovascular morbidity (due to non-fatal ischemic heart events and heart decompensation) in men with prostate cancer in two large studies, though cardiovascular mortality was notably not increased.^[40][100]

In addition to the liver, ethinylestradiol shows disproportionate estrogenic effects in the uterus.^[7][43][101] This is due to its inability to be inactivated by uterine 17β-hydroxysteroid dehydrogenase (17β-HSD).^[7][43][101] Because of its disproportionate effects in the uterus, ethinylestradiol is associated with a significantly lower incidence of vaginal bleeding and spotting than is estradiol, particularly in combination with progestogens (which induce 17β-HSD expression and hence estradiol metabolism in the uterus),^[7] and is an important contributing factor in why ethinylestradiol, among other reasons and in spite of its inferior safety profile, has been widely used in oral contraceptives instead of estradiol.^[102][103] Although ethinylestradiol has increased effects in the uterus relative to estradiol, it is similarly not associated with an increase in the risk of endometrial hyperplasia and endometrial cancer when used in combination with a progestogen, but instead with a significant decrease.^[7][104]

Oral administration

Estradiol and estrone levels following a single 2 mg dose of oral estradiol in postmenopausal women.^[130]

Mean estradiol levels during 1 to 8 mg per day oral estradiol therapy in transgender women.^[50]

Percent change in estradiol, estrone, LH, and FSH levels over a 24-hour period following a single dose of 2 mg oral estradiol in women.^[131][132]

Levels of estradiol (E2), estrone (E1), and estrone sulfate (E1S) following a single 2 mg oral dose of micronized estradiol in postmenopausal women.^[133] Peak levels of estradiol, estrone, and estrone sulfate were around 35 pg/mL, 300 pg/mL, and 12,000 pg/mL, respectively.^[133]

Estradiol is rapidly and completely absorbed with oral administration.^[134][9] This is true for oral doses of 2 mg and 4 mg, but absorption was found to be incomplete for an oral dose of 8 mg (which showed 76% of the expected bioavailability, based on dose proportionality and area-under-the-curve levels).^[134][135] The oral bioavailability of estradiol is very low, and the hormone must be either micronized or conjugated with an ester, as in estradiol valerate or estradiol acetate, to be bioavailable to an extent that is clinically useful.^{[136][137][138]} This is because estradiol is extensively metabolized during the first pass through the intestines and liver, and micronization increases the rate of absorption and improves the metabolic stability of estradiol.^[134] As micronization is required for significant bioavailability, all oral estradiol tablets are micronized.^[137] The absolute bioavailability of oral micronized estradiol is approximately 5%, with a possible range of 0.1% to 12%.^[1][136] In accordance, the circulating levels of estradiol with 2 mg/day oral estradiol and 100 µg/day transdermal estradiol are similar, in spite of a 20-fold difference in dosage.^[139][9] There is high interindividual variability in the levels of estradiol achieved with oral estradiol, which is likely related to the high first-pass effect.^[9] This variation has been reported to be in the range of 28 to 127% in terms of mean area-under-the-curve levels of estradiol.^[9]

In postmenopausal women, a dosage of 1 mg/day oral micronized estradiol has been found to produce circulating concentrations of 30 to 50 pg/mL estradiol and 150 to 300 pg/mL estrone, while a dosage of 2 mg/day has been found to result in circulating levels of 50 to 180 pg/mL estradiol and 300 to 850 pg/mL estrone.^[43] A study of oral micronized estradiol in transgender women found that mean estradiol levels across a dosage range of 1 to 8 mg/day were almost 50 pg/mL at 1 mg/day, almost 100 pg/mL at 4 mg/day, and just above 150 pg/mL at 8 mg/day, with a wide degree of variation.^[50] A single 10 mg dose of micronized estradiol or estradiol valerate has been found to produce circulating levels of estradiol of about 250 pg/mL in postmenopausal women.^[101] A study that used high- to very high-dose oral micronized estradiol to treat postmenopausal women with ER-positive breast cancer found that mean steady-state estradiol levels in the 6 mg/day group were 302 pg/mL and in the 30 mg/day group were 2,403 pg/mL.^[140]

When taken orally, about 95% of a dose of estradiol is metabolized in the intestines and liver into estrone and estrogen conjugates such as estrone sulfate, estrone glucuronide, and estradiol sulfate, among others, prior to entering the circulation.^{[134][141][142][143]} As a result, circulating estrone and estrogen conjugate levels are markedly elevated, in a highly unphysiological manner, with oral estradiol.^[141][139] Whereas the ratio of circulating estradiol to estrone is about 1:1 in premenopausal women and with transdermal estradiol, oral estradiol produces a ratio of about 1:5 on average and as high as 1:20 in some women.^{[1][7][144][135]} In addition, whereas levels of estradiol with menopausal replacement dosages of oral estradiol are in the range of the follicular phase of the normal menstrual cycle, levels of estrone resemble those during the first trimester of pregnancy.^[145][146] Moreover, whereas normal estrone sulfate levels are 10 to 25 times higher than those of estradiol and estrone in premenopausal women,^[147] levels of estrone sulfate with oral estradiol are an additional 10 to 20 times higher than normal premenopausal estrone sulfate levels.^[139][148] One study found that estrone sulfate levels were 200-fold higher than estradiol levels with 2 mg/day oral micronized estradiol or oral estradiol valerate, and estrone sulfate levels can reach up to nearly 1,000-fold higher concentrations than estradiol in some cases.^[7][9] In contrast to oral estradiol, due to the lack of the first pass, an excess in estrone and estrogen conjugate levels does not occur with transdermal estradiol or other parenteral estradiol routes.^[141][139]

The transformation of estradiol into estrone and estrogen conjugates is reversible, and these metabolites hence can be converted back into estradiol.^[7] About 15% of orally administered estradiol is transformed into estrone and 65% into estrone sulfate.^[9] About 5% of estrone and 1.4% of estrone sulfate can be converted back into estradiol.^[9] An additional 21% of estrone sulfate can be converted into estrone, whereas the transformation of estrone into estrone sulfate is approximately 54%.^[9] The interconversion between estradiol and estrone is mediated by 17β-hydroxysteroid dehydrogenases (17β-HSDs),^[9] whereas the conversion of estrone into estrone sulfate is mediated by estrogen sulfotransferases (ESTs) and the transformation of estrone sulfate into estrone by steroid sulfatase (STS).^[149][150] The metabolic clearance rates and hence blood half-lives of estrogen conjugates like estrone sulfate are far longer than those of estradiol and estrone.^[7][9][139] Estrogen conjugates, primarily estrone sulfate, serve as a large circulating reservoir for estradiol, and because of this, they function to greatly extend the biological half-life of oral estradiol.^[7][9] As such, the biological half-life of oral estradiol is a composite parameter that is dependent on interconversion between estradiol and estrogen conjugates, as well as on enterohepatic recirculation.^[9] Whereas the biological half-life of estradiol given by intravenous injection is only about 1 to 2 hours, the biological half-life of oral estradiol has a range of 13 to 20 hours due to the large and long-lasting pool of estrogen conjugates that is formed during first-pass metabolism and that serves to continuously replenish circulating estradiol levels.^[9][7]

In contrast to estradiol, estrone has very low activity as an estrogen.^{[7][151][7][12]} The affinities of estrone for the human ERs and its estrogenic activity have been reported to be approximately 3 to 4% of those of estradiol.^[7] In addition, unlike estradiol and estriol, estrone is not accumulated in target tissues.^[7] Because estrone can be transformed into estradiol, most of its activity in vivo is actually due to conversion into estradiol.^[7] In accordance, dosages of oral and transdermal estradiol that achieve similar levels of estradiol have been found, in spite of markedly elevated levels of estrone with oral estradiol but not with transdermal estradiol, to possess equivalent and non-significantly different potency in terms of clinical measures including suppression of LH and FSH levels, inhibition of bone resorption, and relief of menopausal symptoms such as hot flashes.^{[7][141][128][152]} In addition, estradiol levels were found to correlate with these effects, while estrone levels did not.^[141][128] These findings suggest that estrone contributes very little or not at all to the estrogenic potency of estradiol, while also not antagonizing the estrogenic activity of estradiol.^{[7][141][128][152]} This contradicts some in vitro research suggesting that estrone might be able to partially antagonize the actions of estradiol.^{[153][154][155]}

Distribution of 17β-HSD activities for formation of estradiol (E2) versus formation of estrone (E1) in human tissues.^[156][8]

Distribution of STS and EST activities for interconversion of estrone (E1) and estrone sulfate (E1S) in adult human tissues.^[157]

Relative activation and inactivation of estradiol from and to the circulating estrone and estrone sulfate pool in tissues throughout the body

On the other hand, it has been suggested that the high levels of estrone and/or estrone conjugates with oral estradiol may result in excessive estradiol levels in certain tissues such as the breasts and endometrium due to high expression of the requisite enzymes (17β-HSDs and STS) necessary to transform these metabolites back into estradiol in these tissues.^{[146][139][131][158]} In accordance, circulating levels of estrone sulfate have been found to be positively associated with breast density in postmenopausal women treated with oral estradiol, with a 1.3% increase in breast density observed for every 1 ng/mL increase in estrone sulfate levels.^[159][160] Similarly, levels of estradiol, estrone, and estrone sulfate are all strongly associated with the risk of breast cancer in women.^[159] Preclinical studies have shown that estrone sulfate, via local transformation into estradiol, stimulates the growth of mammary cancer cells.^[161][162] In addition, a study found that administration of estrone sulfate was more efficient in delivering free estradiol into mammary cancer cells and increasing mammary tumor volume than was administration of estradiol itself.^[161]

Due to the first pass through the liver, disproportionate and supraphysiological levels of estrogens occur locally in the liver with oral estradiol.^[89][9] These levels are approximately 4- to 5-fold higher than in the circulation.^[89] As a result, there is abnormally high estrogenic signaling in the liver with oral estradiol, and a variety of unphysiological effects on liver protein synthesis result.^[7][9] Via modulation of liver protein synthesis, oral estradiol increases the risk of blood clots,^[163] suppresses growth hormone (GH)-mediated insulin-like growth factor 1 (IGF-1) production,^[164][165] increases circulating levels of a variety of binding proteins including thyroid binding globulin (TBG), cortisol binding globulin (CBG), sex hormone binding globulin (SHBG), growth hormone binding protein (GHBP),^[166] insulin-like growth factor-binding proteins (IGFBPs),^[167] and copper binding protein (CBP),^[142][168] and produces positive blood lipid changes, among a variety of other effects.^{[143][169][9]} In contrast to oral estradiol, transdermal estradiol has minimal to no impact on liver protein synthesis.^[7] As an example, a study found that 1 mg/day oral estradiol significantly increased SHBG levels by 45%, while 50 µg/day transdermal estradiol increased SHBG levels non-significantly by only 12%.^[90][91][92]

In the circulation, approximately 38% of estradiol is reversibly bound to SHBG and 60% is reversibly bound to albumin in women under normal physiological circumstances, with 2 to 3% of total estradiol circulating free or unbound at any given time.^{[134][136][1]} Only estradiol that is free or unbound is able to be enter target cells and hence is biologically active.^{[1][9]:249[112]} The increase in SHBG levels with oral estradiol (e.g., +50%) can result in a clinically meaningful increase in the fractions of sex hormones like estradiol and testosterone that are bound to SHBG, whereas this is not the case with typical clinical dosages of transdermal estradiol.^[170][112] The increase in the fraction of estradiol bound to SHBG results in a significant decrease in the percentage of free or unbound and hence bioactive estradiol.^[1][112] As a result, the bioavailability and potency of oral estradiol may be diminished relative to parenteral estradiol routes by some amount.^[112][1]

Buccal administration

Estradiol has been studied for use by buccal administration.^{[7][171][172][173]}

Sublingual administration

Estradiol levels over a 24-hour period following a single dose of different doses of sublingual (s.l.) and oral (p.o.) estradiol in postmenopausal women.^[174]

Percent change in estradiol, estrone, LH, and FSH levels over an 11-hour period following a single dose of 0.5 mg sublingual estradiol in postmenopausal women.^[119]

Micronized estradiol tablets can be taken sublingually instead of orally.^[175] All estradiol tablets are micronized, as estradiol cannot be absorbed efficiently otherwise.^[137] Sublingual ingestion bypasses first-pass metabolism in the intestines and liver.^[176] It has been found to result in levels of estradiol and an estradiol-to-estrone ratio that are substantially higher in comparison to oral ingestion.^[174] Maximal circulating levels of estradiol are as much as 10-fold higher with sublingual administration relative to oral administration, and the absolute bioavailability of estradiol is approximately 5-fold higher.^[7] On the other hand, levels of estradiol fall rapidly with sublingual administration, whereas they remain elevated for an extended period of time with oral administration.^[7][9] This is responsible for the divergence between the maximal estradiol levels achieved and the absolute bioavailability.^[7][9]

The rapid and steep fall in estradiol levels with sublingual administration is analogous to the case of intravenous administration of the hormone, in which there is a rapid distribution phase of 6 minutes and terminal disposition phase of only 1 to 2 hours.^[7][9][2] In contrast to intravenous and sublingual administration, the terminal half-life of estradiol with oral administration is 13 to 20 hours.^[7][9] The difference is due to the fact that, upon oral administration, a large hormonally inert pool of estrogen sulfate and glucuronide conjugates with extended terminal half-lives is reversibly formed from estradiol during first-pass metabolism, and this pool serves as a metabolism-resistant and long-lasting circulating reservoir for slow reconversion back into estradiol.^[7][9]

Upon sublingual ingestion, a single 0.25 mg tablet of micronized estradiol has been found to produce peak levels of 300 pg/mL estradiol and 60 pg/mL estrone within 1 hour.^[7] A higher dose of 1 mg estradiol was found to result in maximum levels of 450 pg/mL estradiol and 165 pg/mL estrone.^[7] This was followed by a rapid decline in estradiol levels to 85 pg/mL within 3 hours, whereas the decline in estrone levels was much slower and reached a level of 80 pg/mL after 18 hours.^[7]

Although sublingual administration of estradiol has a relatively short duration, the drug can be administered multiple times per day in divided doses to compensate for this.^[7] In addition, it is notable that the magnitude of the genomic effects of estradiol (i.e., signaling through the nuclear ERs) may, at least in some cases, be dependent on the total exposure as opposed to the duration of exposure.^[7] For instance, in normal human epithelial breast cells and ER-positive breast cancer cells, the rate of breast cell proliferation has been found not to differ with estradiol incubation of 1 nM for 24 hours and incubation of 24 nM for 1 hour.^[7] In other words, short-term high concentrations and long-term low concentrations of estradiol appear to have the same degree of effect in terms of genomic estrogenic signaling, at least in terms of breast cell proliferation.^[7]

On the other hand, non-genomic actions of estradiol, such as signaling through membrane estrogen receptors like the GPER, may be reduced with short-term high concentrations of estradiol relative to more sustained levels.^[7] For instance, although daily intranasal administration of estradiol (which, similarly to sublingual administration, produces extremely high peak levels of estradiol followed by a rapid fall in estradiol levels) is associated in postmenopausal women with comparable clinical effectiveness (e.g., for hot flashes) relative to longer acting routes of estradiol administration, it is also associated with significantly lower rates of breast tension (tenderness and enlargement) relative to longer acting estradiol routes, and this is thought to reflect comparatively diminished non-genomic signaling.^[7]

The effects of sublingual estradiol on gonadotropin levels have been studied in postmenopausal women.^[119]

Intranasal administration

Intranasal administration of estradiol has been studied.^[171][7]

Estradiol is or was available as a nasal spray (brand name Aerodiol) in some countries.^{[177][178][179][180]} The Aerodiol product was discontinued in 2007.^[181][182] Intranasal estradiol has pharmacokinetics similar to those of sublingual estradiol, including a sharp peak in levels followed by a rapid decline in concentrations.^[7] Despite the relatively short duration of intranasal estradiol, it is has similar effectiveness to other, longer-lasting routes of administration in terms of relief of menopausal symptoms like hot flashes.^[7]

Transdermal administration

Levels of estradiol at steady-state over a period of 4 days with different dosages of Vivelle-type (Vivelle, Vivelle-Dot, Mylan generic) twice-weekly transdermal estradiol matrix patches applied to the abdomen and worn until day 4 in postmenopausal women.^{[183][184][185]}

Levels of estradiol over a period of 7.5 days after a single application of different dosages of a Climara-type (Climara, Menostar, Mylan generic) once-weekly transdermal estradiol matrix patch to the abdomen and removed on day 7 in postmenopausal women.^{[186][187][188]}

Levels of estradiol and estrone with once daily appli­cation of 1.25 g of a transdermal estradiol gel (EstroGel) containing 0.06% or 0.75 mg estradiol after 14 days of continuous therapy in postmenopausal women.^[189]

Levels of estradiol and estrone with application of a single 50 µg/day estradiol transdermal reservoir patch (Estraderm) in postmenopausal women.^[190]

Levels of estradiol over the course of 15 days with a single application of a 100 µg/day estradiol transdermal reservoir patch (Estraderm) in postmenopausal women.^[112] This patch has a 3- to 4-day duration and is designed for twice-weekly use. The patch was applied on day 1 and was removed on day 7.^[112] In one group, ethanol was injected into the area between the patch and the skin on day 3 to restore the solvent, and resulted in increased bioavailability and a prolonged duration of the patch.^[112]

Transdermal estradiol is available in the forms of patches, gels, emulsions, and sprays.^{[191][192][7][112]} In the case of gels, emulsions, and sprays, the route is sometimes referred to as topical rather than as transdermal.^{[192][190][3]} Estradiol patches have an extended duration and are available for twice-weekly (3–4-day) and once-weekly (7-day) application, while gels, emulsions, and sprays are administered daily.^{[192][43][7][193]} There are two types of estradiol patches: reservoir patches, which have been described as first-generation patches, and matrix patches, which are considered to be improved second-generation patches.^[7][9][192] Reservoir patches were designed for twice-weekly application, while matrix patches have been produced for both twice-weekly and once-weekly application.^[9] Reservoir patches of estradiol (e.g., Estraderm) are mostly no longer used, with most estradiol patches available today being matrix patches (e.g., Alora, Climara, Esclim, Estradot, FemPatch, Menostar, Oesclim, Vivelle, and Vivelle-Dot).^[192] When estradiol is administered as a hydroalcoholic gel such as EstroGel, it dries within 2 to 5 minutes following application to the skin.^[189]

Estradiol has moderate skin permeability, which is based on the lipophilicity and hydrophilicity of a compound.^[7] In general, the more hydroxyl groups that are present in a steroid, the less its skin permeability.^[7] For this reason, estrone and progesterone have high skin permeability, while estriol and cortisol have low skin permeability.^[7] Regardless of administration form, such as patch or gel, transdermal estradiol is transported into the skin, including through the stratum corneum, epidermis, and dermis, by a passive diffusion process.^[7][189] Following this, estradiol is then taken up by local capillary blood vessels and delivered into the circulation.^[7] There is a depot effect in the skin with transdermal estradiol, which results in continuous delivery of transdermal estradiol into the circulation.^[112][189] This is because the skin functions as a semipermeable membrane and there is a concentration gradient between the application site of transdermal estradiol and capillary blood, with the rate of diffusion of estradiol across the stratum corneum being the specific rate-limiting factor in absorption.^[7][189] As a result, peaks and troughs in circulating estradiol levels are limited, and the skin and subcutaneous fat act as a reservoir of estradiol that maintains circulating estradiol levels between doses.^[112] For these reasons, transdermal estradiol can provide near-constant circulating levels of estradiol, similarly to oral estradiol.^[112][7] A single application of a transdermal estradiol gel (e.g., EstroGel) results in a sustained increase in estradiol levels for at least 24 hours.^[112][189] Enzymes that metabolize estradiol are minimally expressed in the skin, and for this reason, the metabolism of estradiol in the skin is low.^[7] The apparent elimination half-life of estradiol with transdermal estradiol gel (as EstroGel) is 36 hours.^[189]

Transdermal estradiol bypasses the intestines and liver and hence the first-pass metabolism that is associated with oral administration.^[7] As a result, it has much greater bioavailability and potency than oral estradiol.^[7] In spite of a 20-fold difference in dosage, the circulating levels of estradiol with 2 mg/day oral estradiol and 100 µg/day transdermal estradiol are similar.^[139][9] A dosage of 1 mg/day oral estradiol is considered to be roughly equivalent to 50 µg/day transdermal estradiol and a dosage of 2 mg/day oral estradiol is considered to be equivalent to 100 µg/day transdermal estradiol.^[148][9][7] In addition, unlike oral estradiol, transdermal estradiol is not associated with supraphysiological concentrations of estrone or estrogen conjugates like estradiol sulfate, and transdermal estradiol does not have disproportionate effects on liver protein synthesis.^[7] In accordance, estradiol patches, at typical menopausal replacement dosages, have been found not to increase the risk of blood clots or insulin resistance,^[163][9] nor to affect hepatic SHBG, IGF-1, GHBP,^[166] IGFBP,^[167] and other protein production and by extension circulating hepatic protein levels.^{[164][165][168]}

Transdermal administration of estradiol via patch or gel produces an estradiol to estrone ratio of about 1:1.^[7][9] Estradiol patches delivering a daily dosage of 0.05 mg (50 µg) achieve mean estradiol and estrone levels of 30 to 65 pg/mL and 40 to 45 pg/mL, respectively, while a daily dosage of 0.1 mg (100 µg) attains respective mean levels of 50 to 90 pg/mL and 30 to 65 pg/mL of estradiol and estrone.^[43] Once daily application of 1.25 g topical gel containing 0.75 mg estradiol (brand name EstroGel) for 2 weeks was found to produce mean peak estradiol and estrone levels of 46.4 pg/mL and 64.2 pg/mL, respectively.^[189] The time-averaged levels of circulating estradiol and estrone with this formulation over the 24-hour dose interval were 28.3 pg/mL and 48.6 pg/mL, respectively.^[189] Levels of estradiol and estrone are stable and change relatively little over the course of the 24 hours following an application, indicating a long duration of action of this route.^[189] Steady-state levels of estradiol are achieved after 3 days of application.^[189] A higher dosage of estradiol gel containing 1.5 mg estradiol per daily application has been found to produce mean estradiol levels of 40 to 100 pg/mL and estrone levels of 90 pg/mL, while 3 mg per day has been found to result in respective mean estradiol and estrone levels of 60 to 140 pg/mL and 45 to 155 pg/mL.^[43] Following removal of an estradiol patch, circulating estradiol levels decrease to baseline within 24 hours.^[7]

Typical dosages of estradiol patches are intended to provide the minimum amount of estrogen replacement necessary for the effective alleviation of menopausal symptoms, and for this reason, they achieve relatively low levels of estradiol.^[7] A dosage of two to six 100 µg/day transdermal estradiol patches can achieve mean levels of estradiol in the area of 200 to 400 pg/mL and can be used as a form of high-dose estrogen therapy, for instance to suppress testosterone levels in the treatment of prostate cancer in men and in feminizing hormone therapy for transgender women.^[11][47][65]

Transdermal estradiol patches are described as delivering a fixed amount of estradiol such as 50 µg/day or 100 µg/day.^[7] However, there is large interindividual variability and intraindividual variability`in the pharmacokinetic parameters of transdermal estradiol, and fluctuations in circulating estradiol levels with estradiol patches is almost as great as with oral estradiol.^[7][9][112] As such, the actual delivery rate of estradiol and mean levels of estradiol achieved with transdermal estradiol patches may be different from what is described and from the mean levels observed in clinical studies, respectively.^[7] A wide range of estradiol levels are measured in women using the same estradiol patch or gel and dosage, with an up to 10-fold difference in levels.^[7][112] For example, the interindividual variability with Estraderm reservoir patches ranges from 25 to 225%.^[112] In as many as 30% of women treated with a 50 µg/day estradiol patch, estradiol levels are low.^[7] There are also significant short-term intraindividual differences in estradiol levels with estradiol patches; estradiol levels can fluctuate considerably from hour to hour.^[7][190] In addition, estradiol levels with estradiol patches are higher in the evening than in the morning, which may be due to circadian variations in skin blood flow that may influence absorption.^[7] In terms of area-under-the-curve levels of estradiol achieved, the interindividual variability of transdermal estradiol has been found to be 20 to 44% using different transdermal formulations, and the intraindividual variability with transdermal estradiol has been found to be 20%.^[9] Factors which may contribute to inter- and intraindividual variability with transdermal estradiol include skin location and thickness; hair follicle density; solvent (alcohol) evaporation; skin dehydration, ambient temperature, and humidity; and reservoir size.^[112]

Estradiol patches are associated with local skin reactions and such as irritation in 14.2% of individuals (with reservoir patches), mild-to-moderate erythema (redness) in 50 to 60% of individuals, and allergic reactions due to cutaneous sensitization.^[7][9] Up to 5% of people using reservoir patches may discontinue therapy due to skin reactions.^[9] Visible adhesive residues are also often left by estradiol patches following their removal.^[7]

Vaginal administration

Percent change in estradiol, estrone, LH, and FSH levels over a 24-hour period following a single dose of 1 mg vaginal micronized estradiol in women.^[131]

Vaginal estradiol is available in the forms of tablets, creams, inserts or suppositories, and rings.^{[192][7][191]} Vaginal estradiol tablets, creams, and inserts are usually administered once daily to twice weekly, whereas vaginal estradiol rings have a sustained action and are replaced once every 90 days.^[192][7] Vaginal estradiol can be used both as a systemic form of estradiol therapy, and at very low doses to selectively achieve a local vaginal effect without systemic effects, for instance in the treatment of menopausal symptoms such as vaginal atrophy and dryness.^[7][213]

Vaginal estradiol is rapidly and almost completely absorbed.^[171] The absorption of vaginal estradiol is slightly greater in women with vaginal atrophy.^[171] Similarly to transdermal estradiol, vaginal estradiol has greatly increased potency compared to oral estradiol, with about 10- to 20-fold the comparative potency of oral estradiol.^[7] The greater potency of vaginal estradiol relative to oral estradiol is due to the lack of the first pass associated with oral estradiol and due to local metabolism of estradiol in the vagina being low.^[7] Because of the high estradiol levels achieved, LH and FSH are more strongly suppressed with vaginal estradiol than with other routes.^[171]

A daily dosage of 0.5 mg vaginal micronized estradiol has been found to result in estradiol and estrone levels of 250 pg/mL and 130 pg/mL, respectively.^[43] Vaginal estradiol has a much higher estradiol-to-estrone ratio in comparison to oral estradiol.^[7] The average ratio of estradiol to estrone with vaginal estradiol is 5:1, compared to 1:5 in the case of oral estradiol, a 10-fold difference.^[7]

As vaginal estradiol is not subject to a first pass and bypasses the intestines and liver, it does not affect liver protein synthesis at menopausal replacement dosages, similarly to transdermal estradiol.^[214]

Rectal administration

Estradiol has been studied for use by rectal administration.^{[215][216][217][218][219]} Rectal administration of estradiol is described as qualitatively and quantitatively similar to vaginal administration of estradiol.^[216][171] Lauritzen (1986) reported that 3 hours after a rectal dose of 1 mg micronized estradiol, estradiol levels increased by 620 pg/mL and estrone levels increased by 120 pg/mL.^[216][171] Subsequently, Lauritzen (1990) reported that 0.5 mg/day and 1 mg/day rectal estradiol resulted in respective estradiol levels of 363 pg/mL and 515 pg/mL 6 hours after the last dose.^[215] These estradiol levels are fairly similar to those achieved by vaginal estradiol.^{[216][215][171]} The estradiol-to-estrone ratio of rectal estradiol is about 5:1, which is the same as that of vaginal estradiol.^[216][171] Absorption of rectal estradiol occurs rapidly within 30 to 60 minutes, maximal estradiol levels occur at 3 hours post-dose, and circulating estradiol levels are reportedly maintained for 4 to 10 hours.^[216][171] The duration of rectal estradiol is said to necessitate administration 1 to 2 times per day.^[216] Irritation of the intestines does not usually occur.^[216] Rectal administration of estradiol is not accepted by all individuals.^[216]

Rectal estradiol has been studied as a form of high-dose estrogen therapy for the treatment of prostate cancer in men.^[218]

Rectal administration of estriol, which has similar properties to estradiol, has also been studied.^[220] Administration of a rectal suppository containing 100 mg estriol resulted in estriol levels in pregnant women at term increasing by about 53%.^[220] Estriol levels at term are normally between 5,000 and 20,000 pg/mL.^{[221][222][223]}

Intramuscular injection

Estradiol levels following single intramuscular injections of 5 mg of different estradiol esters in oil in women.^[125] The dashed line at 0.5 days marks when the injections were given.^[125]

Idealized curves of estradiol levels over a period of 30 days after injection of different estradiol esters in women.^[224] Four data points were used to generate the curves (day 0, peak day, a third day, and day 30).^[224] The measurements from which the points were drawn were taken at 24-hour intervals.^[224] The estradiol esters were given mostly as combined injectable contraceptives together with a progestin.^[224]

Estradiol levels following a single intramuscular injection of 10 mg estradiol valerate or 100 mg estradiol undecylate.^[126] The solid lines are the average estradiol levels and the dashed lines are the highest and lowest observed levels, to show interindividual variability.^[126]

Estradiol levels with intramuscular injection of different dosages of polyestradiol phosphate once every 4 weeks for 6 months in men.^[10]

Estradiol, in an ester prodrug form such as estradiol valerate or estradiol cypionate, can be administered by intramuscular injection, via which a long-lasting depot effect occurs.^[125][6] In contrast to the oral route, the bioavailability of estradiol and its esters like estradiol valerate is complete (i.e., 100%) with intramuscular injection.^[2] The levels of estradiol that are achieved with typical clinical dosages of injections are very high compared to other routes.^{[7][111][225][125][10]}

A single 4 mg intramuscular injection of estradiol cypionate or estradiol valerate has been found to result in maximal plasma levels of estradiol of about 250 pg/mL and 390 pg/mL, respectively, with levels declining to 100 pg/mL (the baseline for estradiol cypionate) by 12 to 14 days.^[111][225] A single 2.5 mg intramuscular injection of estradiol benzoate in patients being administered a GnRH analogue (and hence having minimal baseline levels of estrogen) was found to result in serum estradiol levels of >400 pg/mL at 24 hours post-administration.^[125] The differences in the serum levels of estradiol achieved with these different estradiol esters may be explained by their different rates of absorption, as their durations and levels attained appear to be inversely proportional.^[125] For instance, estradiol benzoate, which has the shortest duration (4 to 5 days with a single intramuscular injection of 5 mg), produces the highest levels of estradiol, while estradiol cypionate, which has the longest duration (~11 days with 5 mg), produces the lowest levels of estradiol.^[125] Estradiol valerate was found to have a duration of 7 to 8 days after a single intramuscular injection of 5 mg.^[125]

A study of combined high-dosage intramuscular estradiol valerate and hydroxyprogesterone caproate in peri- and postmenopausal and hypogonadal women (a pseudopregnancy regimen), with specific dosages of 40 mg weekly and 250 mg weekly, respectively, was found to result in serum estradiol levels of 3,028 to 3,226 pg/mL after three months and 2,491 to 2,552 pg/mL after six months of treatment from a baseline of 27.8 to 34.8 pg/mL.^[226]

Polyestradiol phosphate is an ester prodrug of estradiol in the form of a polymer which is used via intramuscular injection primarily to treat prostate cancer, but also to treat breast cancer and menopausal symptoms.^[227][228] It has an extremely long duration of action, with a terminal half-life of about 70 days (10 weeks) following a single intramuscular administration of the medication.^[46] In addition, unlike most other estradiol esters, the estradiol levels achieved with polyestradiol phosphate are highly constant and uniform.^[46] Levels of estradiol in men with intramuscular injections of polyestradiol phosphate once every 4 weeks were about 350 pg/mL with 160 mg, 450 pg/mL with 240 mg, and almost 700 pg/mL with 320 mg, all measured after 6 months of treatment.^[10] Polyestradiol phosphate has been discontinued in many countries, and remains available today in only a few countries, mostly the Nordic countries of Europe.^[228][229]

Estradiol is available in the form of an aqueous suspension of 1.0 mg estradiol encapsulated in microspheres for use by intramuscular injection once a month under the brand name Juvenum E in Mexico.^[230][231] It achieves circulating estradiol levels of 163 pg/mL to 219 pg/mL in the first 3 to 12 hours following injection, which decrease to 42.4 to 65.7 pg/mL during the first 4 days post-injection and to 20 to 35 pg/mL after 8 days, with levels remaining in this range thereafter over 30 days.^[230] These estradiol levels are similar to the normal levels that occur during the early follicular phase of the menstrual cycle in premenopausal women (24 to 75 pg/mL).^[230] The elimination of the formulation follows three phases: a rapid phase in the first 2 days, a second phase during days 2 to 12 days with a biological half-life of 7.2 to 9.9 days, and a third phase in which estradiol levels remain elevated above baseline for up to 30 days.^[230]

Subcutaneous injection

Estradiol levels after subcutaneous (s.c.) or intramuscular (i.m.) injection of 5 mg estradiol cypionate in aqueous suspension.^[6]

Estradiol esters like estradiol valerate and estradiol cypionate can be given by subcutaneous injection instead of intramuscular injection.^[232] Subcutaneous and intramuscular injection of estradiol cypionate in an aqueous suspension has been found to result in levels of estradiol and other pharmacokinetic parameters (e.g., duration) that were virtually identical.^[6] However, subcutaneous injections may be easier and less painful to perform compared to intramuscular injections, and hence may result in improved compliance and satisfaction.^[6] Studies have shown that subcutaneous injection of closely related steroid esters in oil like the androgen esters testosterone cypionate, testosterone enantate, and nandrolone decanoate is effective and has similar pharmacokinetics to intramuscular injection as well.^{[233][65][234][235][236][237][238]} In addition, studies have found that many intramuscular injections are really subcutaneous injections, as people often do not actually penetrate deep enough to inject into muscle.^[239][240] This is particularly prevalent with injections into the buttocks and in overweight and obese individuals, due to the thicker fat layer over muscle.^[239][240]

Subcutaneous implant

Levels of estradiol over a period of 14 months following surgical implantation of a subcutaneous pellet of 100 mg estradiol in women.^[7]

Estradiol can be administered in the form of a very long-lasting subcutaneous pellet implant.^[7][241] These implants are replaced once every 6 to 12 months, and can achieve high and very constant circulating levels of estradiol.^{[7][242][243]} They are surgically inserted by a trained physician in a medical office or clinic and can be placed into locations including the lower abdomen, lower back, buttocks, or hips.^{[7][242][241]} Subcutaneous implants containing 25, 50, and 100 mg estradiol for replacement usually once every 6 months are available as approved pharmaceutical medications.^[243] Pharmaceutical estradiol implants have been said to be used almost exclusively in the United Kingdom.^[244] An estradiol implant has not been approved by the FDA as a pharmaceutical medication in the United States, but hormone implants, including estradiol implants, are available as custom compounded products in this country.^{[245][246][247]}

Estradiol implants are advantageous in that some women seem to need higher levels of estradiol for adequate relief of menopausal symptoms, and subcutaneous implants are easily able to achieve such levels.^[243][7] Conversely, this is not necessarily the case with oral or transdermal estradiol.^[243][7] Another major advantage of estradiol implants is convenience and guaranteed compliance.^[243] They also do not have the issues pertaining to first-pass metabolism and liver protein synthesis of oral estradiol.^[243][7] A major disadvantage of estradiol implants is that they cannot be easily removed should this be necessary.^[243] There are also concerns about accumulation of estradiol levels with long-term repeated pellet implantation.^[243][7] Estradiol levels may remain above baseline for a year or in some cases 3 to 4 years following the last implant insertion.^[243] During this time, progestogen therapy should be continued to avoid the risk of endometrial changes.^[243][242] Regular monitoring of estradiol levels and adjustment of dosing is recommended during therapy with estradiol implants.^[243]

Tachyphylaxis of relief of vasomotor symptoms, or hot flashes returning even with normal supraphysiological estradiol levels, may occur in a small subset of cases with subcutaneous estradiol implants.^{[243][7][244][248][242]} The reason for this is unknown, but may be a paradoxical effect of the high levels of estradiol achieved and/or a product of receptor desensitization caused by the long-term gradually decreasing levels of estradiol.^[243][7] Such symptoms reportedly do not start to occur until estradiol levels begin to decrease, although there are also reports of them occurring 3 to 16 weeks (1 to 4 months) after implant insertion when levels should still be constant.^[243][7] Hot flashes have notably been reported in pregnant women, who have very high levels of estradiol.^[249] When recurrence of hot flashes occurs with estradiol implants, treated women often complain that their implant has "run out".^[243] Such symptoms can be temporarily offset with the use of oral or transdermal estradiol.^[243]

Following insertion of an estradiol implant, levels of estradiol rapidly increase, remain constant for about 4 months, and then gradually decrease.^[243] A 25 mg subcutaneous estradiol pellet has been found to result in average estradiol levels of 90 pg/mL for 6 months, while two 25 mg pellets (50 mg total) resulted in estradiol levels of 180 pg/mL after 24 hours and levels of 100 to 120 pg/mL for 6 months.^[7] Higher-dose implants resulted in estradiol levels for 50 mg of 100 pg/mL, for 75 mg of 140 pg/mL, and for 100 mg of 150 pg/mL.^[7] Estradiol levels are generally 50% higher than those of estrone, for an estradiol-to-estrone ratio of 1.5:1.^[7] Extremely high levels of estradiol of between 400 to 1,000 pg/mL have been observed in a small subset of women treated with estradiol implants and notably in those experiencing symptoms of tachyphylaxis.^[243][7]

General (ADME)

Metabolism

Metabolic pathway of estradiol in humans[250][251][252] Estradiol Estrone sulfate Estrone glucuronide 2-Hydroxyestrone Estrone 4-Hydroxyestrone 2-Methoxyestrone 16α-Hydroxyestrone 4-Methoxyestrone 17-Epiestriol Estriol 16-Epiestriol 17β-HSD EST STS UGT1A3 UGT1A9 CYP450 CYP450 COMT CYP450 COMT unidentified 17β-HSD unidentified This diagram illustrates the metabolic pathways involved in the metabolism of estradiol in humans. In addition to the transformations shown in the diagram, conjugation (e.g., sulfation and glucuronidation) occurs with estradiol and metabolites that have one or more available hydroxyl (-OH) groups.

There are several major pathways of estradiol metabolism, which occur both in the liver and in other tissues:^[9][7][1]

• Dehydrogenation by 17β-hydroxysteroid dehydrogenase (17β-HSD) into estrone
• Conjugation by estrogen sulfotransferases and UDP-glucuronyltransferases into C3 and/or C17β estrogen conjugates like estrone sulfate and estradiol glucuronide
• Hydroxylation by cytochrome P450 enzymes such as CYP1A1 and CYP3A4 into catechol estrogens like 2-hydroxyestrone and 2-hydroxyestradiol as well as 16-hydroxylated estrogens like 16α-hydroxyestrone and estriol (16α-hydroxyestradiol)

Both dehydrogenation of estradiol by 17β-HSD into estrone and conjugation into estrogen conjugates are reversible transformations.^[9][7] However, in regards to sulfation and desulfation, transformation of estrone into estrone sulfate is predominant relative to the reverse reaction.^[9][157]

Estradiol can also be reversibly converted into long-lived lipoidal estradiol forms like estradiol palmitate and estradiol stearate as a minor route of metabolism.^[8]

The terminal half-life of estradiol administered via intravenous injection is 2 hours in men and 50 minutes in women.^[2] Other routes of administration of estradiol like oral administration or intramuscular injection have far longer terminal half-lives and durations of action due to (1) the formation of a large circulating reservoir of metabolism-resistant estrogen conjugates that can be reconverted back into estradiol and/or (2) the formation of slowly-releasing depots.^[9][7]

The metabolic clearance rates of estradiol, estrone, and estrone sulfate are 580 L/day/m², 1,050 L/day/m², and 80 L/day/m², respectively.^[7]