Equilin Research Articles

PGRMC1 can trigger estrogen-dependent proliferation of breast cancer cells: estradiol vs. equilin vs. ethinylestradiol

Objective: Previous studies have shown that progesterone receptor membrane component 1 (PGRMC1) expressed in breast cancer tissue can predict a worse prognosis for breast cancer patients. Moreover, we demonstrated that PGRMC1 can increase the proliferation of progestogens. However, the role of PGRMC1 in terms of estrogen-induced proliferation and comparing different estrogens is still unclear.Methods: Non-transfected and PGRMC1-transfected T-47D cells were stimulated with estradiol (E2), with equilin (EQ), or with ethinylestradiol (EE) at 1, 10, and 100 nmol/l. Increase of proliferation was compared with a control (without estrogens) and with the estrogen-induced stimulation in empty vector cells vs. PGRMC1-transfected cells.Results: The empty vector cells showed significant proliferation (12–15%) with all three estrogens only at the highest concentration, with no relevant differences between the estrogens. PGRMC1-transfected cells showed about three-fold higher proliferation (29–66%), whereby E2 elicited the strongest and EE the lowest proliferating effects, significantly lower compared to E2 and also compared to EQ. No significant differences were seen between E2 and EQ.Conclusions: PGRMC1 increases strongly the estrogen-dependent breast cell proliferation. The proliferating effects of EE may be lower compared to E2 and EQ. This could have importance in comparing hormone therapy and contraception. Thus, PGRMC1 not only could predict the risk using progestogens but also of different estrogens.

Matrix effect during the membrane-assisted solvent extraction coupled to liquid chromatography tandem mass spectrometry for the determination of a variety of endocrine disrupting compounds in wastewater

Membrane-assisted solvent extraction (MASE) coupled to liquid chromatography–triple quadrupole mass spectrometry (LC–MS/MS) was studied for the determination of a variety of emerging and priority compounds in wastewater. Among the target analytes studied certain hormones (estrone (E1), 17β-estradiol (E2), androsterone (ADT), 17α-ethynyl estradiol (EE2), diethylstilbestrol (DES), equilin (EQ), testosterone (TT), mestranol (MeEE2), 19-norethisterone (NT), progesterone (PG) and equilenin (EQN)), alkylphenols (APs) (4-tert-octylphenol (4tOP), nonylphenol technical mixture (NPs) and 4n-octylphenol (4nOP)) and BPA were included. The work was primarily focused in the LC–MS/MS detection step, both in terms of variable optimization and with respect to the matrix effect study. Both, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) were assessed both in the negative and positive mode, including the optimization of MS/MS operating conditions. The best results were obtained, in most of the cases, for ESI using 0.05% ammonium hydroxide as buffer solution in the mobile phase, composed with methanol and water. Under optimum detection conditions, matrix effect during the detection step was thoroughly studied. Dilution, correction with deuterated analogues and clean-up of the extracts were evaluated for matrix effect correction. Clean-up with Florisil together with correction with deuterated analogues provided the most satisfactory results, with apparent recoveries in the 57–136% range and method detection limits in the low ngL−1 level for most of the analytes. For further validation of the method, two separated extraction procedures, the above mentioned MASE, and conventional solid phase extraction (SPE) were compared during the analysis of real samples and comparable results were successfully obtained for E1, E2, EE2, DES, NT, TT, EQ, PG, BPA, ADT, 4nOP, 4tOP, NPs and EQN.

Differentiating Isobaric Steroid Hormone Metabolites Using Multi-Stage Tandem Mass Spectrometry

Steroid hormones and their metabolites are currently undergoing clinical trials as potential therapeutics for traumatic brain injury (TBI). To support this work, it is necessary to develop improved procedures for differentiating isobaric species in this compound class. Equilin sulfate (E-S), estrone sulfate (E1-S), 17α-dihydroequilin sulfate (ADHE-S), and 17β-dihydroequilin sulfate (BDHE-S) are primary constituents in hormone replacement therapies, such as Premarin, which are among pharmaceuticals being investigated for TBI treatment. The latter three compounds are isomers and can be difficult to differentiate in trace analytical determinations. In this work, a systematic study of the fragmentation of ADHE-S, BDHE-S, E1-S, and E-S under different stages of higher order tandem mass spectrometry (MS(n)) and variation of collision energy, allowed optimization of conditions for distinguishing the isomeric structures. For epimeric variants (e.g., ADHE-S versus BDHE-S; α- versus β-stereoisomerization in the C-17 position), differentiation was achieved at MS(4) and fragmentation was demonstrated through MS(5). Computational analysis was performed to further explore differences in the fragmentation pathways due to changes in stereochemistry.

Photochemical induced changes of in vitro estrogenic activity of steroid hormones

Steroid estrogens are endocrine disrupting contaminants frequently detected in natural waters. Because these estrogens can elicit significant biological responses in aquatic organisms, it is important to study their rates and pathways of degradation in natural waters and to identify whether the transformation products retain biological activity. Photochemical kinetics experiments were conducted under simulated solar light for the hormones 17β-estradiol (E2), 17α-ethinylestradiol (EE2), estrone (E1), equilin (EQ), and equilenin (EQN) under direct and indirect photolysis conditions. All of these hormones were susceptible to direct photodegradation, with half-lives ranging from 40 min for E1 to about 8 h for E2 and EE2. Indirect photolysis experiments with added Suwannee River fulvic acid (SRFA) lead to faster degradation rates for E2, EE2, and EQ. Added SRFA caused slower photodegradation rates for E1 and EQN, indicating that it acts primarily as an inner filter for these analytes. The well-established yeast estrogen screen (YES) was used to measure the estrogenicity of the analytes and their photoproducts. Results of YES assay experiments show that only the direct photolysis of E1 gave estrogenic products. Lumiestrone, the major E1 direct photolysis product, was isolated and characterized. It formed in 53% yield and exhibited moderate estrogenic activity. When photolysed in the presence of perinaphthenone, a potent synthetic sensitizer, E1 degraded via an indirect photolysis pathway and did not produce lumiestrone or any other active products. These results suggest that under typical natural water conditions photochemical reactions of E2, EE2, EQ, and EQN are expected to produce inactive products while E1 will give the estrogenic product lumiestrone in moderate yield.

Application of density functional theory (DFT) to study the properties and degradation of natural estrogen hormones with chemical oxidizers

Estrone (E1), 17β-estradiol (E2), estriol (E3), equilin (EQ) and 17α-estradiol (17α) estrogen hormones are released by humans and animals and have been detected in the environment and municipal wastewater treatment plants. The structural and electronic properties of natural hormone molecules are investigated by performing density functional theory calculations and used to predict their properties and chemical behavior. Quantitative structure property relationship (QSPR) approach is applied to correlate the estrogenicity associated with the natural estrogen hormones according to their molecular properties. The obtained relationship reveals the importance of the frontier molecular orbital energy in the interpretation of estrogenic activity of hormones, which is consistent with the previous research. Moreover, the obtained molecular descriptors also aid determination of the degradability of hormones, and to rationalize degradation pathways, with chemical oxidizers such as ozone and hydroxyl radical. Both types of interactions belong to the orbital-controlled reactions. The active sites determined by Fukui functions for the estrogen hormone molecules confirm the reaction pattern that initiates the attack of the aromatic ring for both ozone and hydroxyl radical. The reactive sites of the molecules are mapped with subsequent reaction intermediates and compared with experimental data obtained from the literature.

Effects of estrogens and selective estrogen receptor modulators on vascular reactivity in the perfused mesenteric vascular bed

Estrogens and selective estrogen receptor modulators (SERMs), such as raloxifene (RAL) and tamoxifen (TAM), acutely relax arteries, but the long-term effects of estrogens and SERMs on vascular reactivity in the mesenteric vasculature have not been well defined. In this study, we used an isolated, perfused mesenteric vascular bed technique to investigate the effect of chronic treatment of estrogens and SERMs on vascular reactivity of the mesenteric bed. Ovariectomized female Sprague-Dawley rats were treated by gavage with vehicle (control, 2-hydroxypropyl-beta-cyclodextrin), ethinyl estradiol, estradiol benzoate, equilin (EQ), TAM, or RAL for 3 wk. EQ and TAM increased vasoconstriction in response to all three vasoconstrictors tested (KCl, norepinephrine, and 5-HT). Ethinyl estradiol increased vasoconstriction in response to KCl and 5-HT, whereas responses to estradiol benzoate and RAL were less consistent. Only EQ (134 +/- 4 mmHg) and TAM (104 +/- 4 mmHg) changed mean arterial blood pressure compared with control (117 +/- 4 mmHg). These data demonstrate that 3-wk gavage treatment with estrogens and SERMs affects vascular reactivity in the mesenteric vascular bed. However, the three formulations of estrogen did not produce equivalent effects, and the effects of the SERMs were different from those of the estrogens.

Separation and detection of the isomeric equine conjugated estrogens, equilin sulfate and Δ 8,9-dehydroestrone sulfate, by liquid chromatography–electrospray-mass spectrometry using carbon-coated zirconia and porous graphitic carbon stationary phases

Equilin-3-sulfate and Δ 8,9-dehydroestrone-3-sulfate are two isomers found in equine conjugated estrogens that differ in structure only by the position of a double bond in the steroid B-ring. These geometric isomers were not resolved on a C 18 column during the analysis of conjugated estrogen drug products by LC–MS using acetonitrile-ammonium acetate buffer as the mobile phase. While no separations of these two isomers were observed on C 18 or other alkyl-bonded silica based phases using a variety of mobile phase conditions, partial separations were achieved on phenyl bonded silica phases with a resolution of 1.5 on a diphenyl phase, and baseline separations were readily achieved on two carbonaceous phases with resolutions routinely exceeding three on graphitic carbon-coated zirconia (Zr-CARB) and resolutions as high as 19 on porous graphitic carbon (Hypercarb). An examination of a selected few conjugated estrogens in the complex drug substance by LC–MS on Hypercarb is presented.

Pharmacokinetics of 17 beta-dihydroequilin sulfate in normal postmenopausal women under steady state conditions.

In the present study, the constant infusion of [3H]17 beta-dihydroequilin sulfate ([3H]17 beta-EqS) was used to estimate the metabolic clearance rate (MCR) of 17 beta-dihydroequilin sulfate (17 beta-EqS) and to measure the conversion of this estrogen to equilin sulfate (EqS), equilenin sulfate (EqnS), 17 beta-dihydroequilenin sulfate (17 beta-EqnS), equilin (Eq), equilenin (Eqn), 17 beta-dihydroequilin (17 beta-Eq), and 17 beta-dihydroequilenin (17 beta-Eqn) in normal postmenopausal women. In seven healthy postmenopausal women, infusion of [3H]17 beta-EqS was started 30 minutes after a priming dose and continued at a constant rate of 10-20 microCi/hour, for 3-6 hours. Three blood samples were taken during and at the end of infusion. From the plasma, unconjugated and sulfate-conjugated estrogens, 17 beta-EqS, EqS, EqnS, 17 beta-EqnS, Eq, Eqn, 17 beta-Eq, and 17 beta-Eqn were isolated and purified by high performance liquid chromatography. The MCR of 17 beta-EqS and the conversion ratios and transfer constants (rho) for precursor (17 beta-EqS) to products were calculated. The mean MCR of 17 beta-EqS was calculated to be 797 +/- 90 L/day or 506 +/- 60 L/m2 per day. The mean conversion ratio (CRPRE-PROBB) was 2.4 +/- 0.4 for EqS, 0.3 +/- 0.04 for EqnS, 0.25 +/- 0.03 for 17 beta-EqnS, 0.09 +/- 0.02 for Eq, 0.03 +/- 0.01 for Eqn, 0.08 +/- 0.02 for 17 beta-Eq, and 0.03 +/- 0.01 for 17 beta-Eqn. In both the sulfate-conjugated and unconjugated forms, the most abundant metabolite formed was Eq. Based on the previously reported MCR of EqS (170 L/m2 per day) and 17 beta-Eq (1252 L/m2 per day), the transfer constants [rho]BB were calculated to be 0.8 +/- 0.10 and 0.20 +/- 0.03, respectively. The results indicate that a large portion of 17 beta-EqS is converted to EqS and the more potent estrogen 17 beta-Eq. The ratio of rho EqS-17 beta-EqS to rho 17 beta-EqS-EqS was calculated to be 0.8 +/- 0.1 and represents the extent of C-17-oxidation and reduction and indicates that substantial amounts of 17 beta-reduced metabolites will still be present in the blood although the oxidation reaction was somewhat greater. The data indicate that, compared with the classic estrogens, the in vivo metabolism of ring B unsaturated estrogens is complex. Thus, although the amount of 17 beta-EqS originally present in the conjugated equine estrogens is small, the pharmacokinetics and pharmacodynamics of EqS, 17 beta-EqS, and the extensive interconversions between these estrogens support the hypothesis that the major in vivo activity of the EqS present in conjugated equine estrogens is expressed through its metabolism to 17 beta-EqS and 17 beta-Eq. Furthermore, the increased estrogenic activity associated with this drug may in part be due to the formation of these 17 beta-reduced metabolites.

Reversed-phase liquid chromatographic method for estrogen determination in equine biological samples.

Equine unsaturated estrogens are the main components of brand formulations indicated for hormonal replacement therapy in both hypogonadic and postmenopausal women. These hormones are produced by the fetoplacental unit during equine gestation. A method is described for the quantitative determination of equilenin (EL), equilin (EQ), 17alpha-dihydroequilin (17dEQ), and estrone (El) in the plasma of a pregnant mare. Blood samples are obtained weekly during pregnancy by jugular venipuncture using sodium ethylenediaminetetracetic as the anticoagulant. For the quantitation of these estrogens, plasma is submitted to enzymatic hydrolysis followed by liquid-liquid extraction. A high-performance liquid chromatographic system equipped with a UV detector set at 220 nm and an ODS Hypersil column is used. The method met precision, specificity, and accuracy requirements. The hormonal levels determined in one target mare throughout pregnancy were 97.91 to 449.13, 116.47 to 266.02, 74.92 to 235.54, and 84.26 to 300.03 ng/mL, reaching a maximum towards the 25th, 20th, 33rd, and 27th weeks, respectively, for E1, EL, EQ, and 17dEQ. The method was successfully tested by quantitating these estrogens in the plasma from a pregnant mare. Its applicability to the study of estrogen bioavailability and bioequivalence is suggested.

Modification of serum lipids and cardiovascular risk by estrogenic active compounds

Cardiovascular disease is an important cause of death among women. After the menopause there is a steep elevation of low-density lipoprotein (LDL) cholesterol and lipoprotein(a) concomitantly with other risk factors ,followed by a striking increase in arteriosclerotic vascular disease ,compatible with a lack of estrogens as a causative factor. The benefit of hormone replacement therapy has been documented in several large studies ,although rigorous randomized trials are still under way. However ,cardiovascular events have been reduced by approximately 50%. In order to optimize beneficial effects and minimize side-effects ,the characteristics of selective estrogenic active compounds are a major target of research. The anti-estrogens are the prototype of partial and selective estrogen functions. The components of conjugated estrogens are under investigation for their specific effects. For example ,both 17 f -dihydroequilin sulfate and 17 f -dihydroequilenin sulfate seem to exert antioxidant activity without significant proliferative effects on uterine or breast tissues. This suggests a selective estrogenic activity dependent on the predominant type of estrogen receptor in the particular tissue. Equilin sulfate exerts a significantly stronger antioxidant activity than 17 g -estradiol in vitro ,and j 8-estrone sulfate increases the lag time for low-density lipoprotein oxidation in vivo . These actions on lipoproteins may add to the quantitative changes of lipoproteins ,while other effects are independent of lipoproteins. The 17 f -dihydroequilin sulfate and equilin sulfate improved the action of insulin in vivo . Data from rhesus monkeys treated with 17 f -dihydroequilenin sulfate indicate that additional mechanisms are probably responsible for the observed cardiovascular protection. Further studies need to be conducted in order to identify selective estrogen receptor modulators and assess their potential ,especially in lowering cardiovascular risk.

Pharmacokinetics and pharmacodynamics of conjugated equine estrogens: chemistry and metabolism.

Conjugated equine estrogens (Premarin), are used extensively for estrogen replacement therapy and prevention of osteoporosis and cardiovascular disease in postmenopausal women. Premarin contains at least 10 estrogens that are the sulfate esters of the ring B saturated estrogens: estrone, 17beta-estradiol, 17alpha-estradiol, and the ring B unsaturated estrogens: equilin, 17beta-dihydroequilin, 17alpha-dihydroequilin, equilenin, 17beta-dihydroequilenin, 17alpha-dihydroequilenin, and delta-8-estrone. Bioassays and estrogen receptor binding studies indicate that all 10 estrogens are biologically active. Moreover, individual components, such as equilin sulfate, delta-8-estrone sulfate, 17beta-dihydroequilin sulfate and estrone sulfate, have potent estrogenic effects. Estrogen sulfates can be absorbed directly from the gastrointestinal tract; however, hydrolysis of the sulfates also occurs in the gastrointestinal tract, and the unconjugated estrogens formed are readily absorbed. After absorption, these estrogens are sulfated rapidly and circulate in this form. The pharmacokinetics of these estrogens indicate that the unconjugated estrogens are cleared from the circulation at a faster rate than their sulfate ester forms. In postmenopausal women, the 17-keto derivatives of these estrogens are metabolized to the more potent 17beta-reduced products. The extent of this activation is nearly 10 times higher with some ring B unsaturated estrogens. The 17beta-reduced metabolites are cleared from the blood at a slower rate than their corresponding 17-keto derivatives. In the human endometrium, equilin is metabolized to 2-hydroxy and 4-hydroxy equilin, with 2-hydroxylation being predominant. In contrast, 2-hydroxy and 4-hydroxy estradiol are formed in equal amounts. Similarly, 16alpha-hydroxylation occurs with both types of estrogens; however, with the ring B saturated estrogens, the 17-keto steroid 16alpha-hydroxy estrone was the major urinary metabolite, whereas with the ring B unsaturated estrogens, the 17beta-reduced steroids, such as 16alpha-hydroxy-17beta-dihydroequilin and 16alpha-hydroxy-17beta-dihydroequilenin, were the major metabolites. This difference in metabolism may be important as it has been suggested that 16alpha-hydroxy estrone (alpha-ketol structure) can form covalent adducts with macromolecules and that it may be oncogenic. These types of interactions will not occur with the 16alpha-hydroxylated-17beta-reduced metabolites of ring B unsaturated estrogens. Since all of the estrogens present in Premarin have estrogenic activity, the pharmacological effects of Premarin are a result of the sum of these individual activities. Therefore, preparations lacking some of these important components may not offer the same degree of beneficial effects as Premarin.

Cardioprotective effects of individual conjugated equine estrogens through their possible modulation of insulin resistance and oxidation of low-density lipoprotein

To examine the independent effects on insulin sensitivity and antioxidative activity of the three most prevalent constituents in Premarin (Wyeth-Ayerst Laboratories, Philadelphia, PA): estrone sulfate (E1S), 50%; equilin sulfate (EqS), 25%, and 17 alpha-dihydroequilin sulfate (17 alpha-ES), 15%. Prospective randomized cross-over study. University of Southern California Medical Center. Eight healthy postmenopausal women, mean age 53 +/- 2 years, and mean body mass index, 26 +/- 2 kg/m2, were enrolled. Each woman received, in randomized succession, daily oral doses of 17 alpha-ES (0.2 mg), E1S (0.625 mg), and EqS (0.3 mg) for 30 days. Oxidation of low-density lipoprotein (LDL) by negatively charged LDL (LDL-) and lag phase duration and measured the plasma glucose disappearance after insulin administration (K(itt)). All three estrogen preparations demonstrated antioxidant effects with E1S demonstrating the most significant changes, followed by EqS and 17 alpha-ES. Using E1S, LDL-levels decreased from a baseline of 3.91 +/- 0.9 to 2.05 +/- 0.32 mg/dL and the lag time increased from 24.5 +/- 6.0 to 87.8 +/- 11.8 minutes. Changes in insulin tolerance tests revealed improved insulin action with the various estrogens. With EqS, K(itt) increased from 3.1% +/- 0.3% to 4.3% +/- 0.3% glucose/min, was intermediate with E1S and was least with 17 alpha-ES. All three conjugated equine estrogens demonstrated antioxidant activity. Also, some improved insulin action was demonstrated. To our knowledge, this is the first in vivo study to examine the effects of these components which may help explain, in part, some of the cardioprotective properties ascribed to Premarin.

Separation of pharmaceutically important estrogens by micellar electrokinetic chromatography

Baseline separation of ten estrogens of the type found in conjugated estrogens tablets from pregnant mares' urine were separated by micellar electrokinetic chromatography with a 20 m M sodium borate-sodium phosphate buffer (pH 8) containing either 75 m M sodium cholate and 15 m M β-cyclodextrin or 50 m M sodium dodecyl sulfate and 20 m M γ-cyclodextrin. The system containing sodium cholate was used to identify estrogens in a pharmaceutical tablet formulation of conjugated estrogens and to determine the amounts and ratio of sodium equilin sulfate to sodium estrone sulfate to confirm tablet conformity with regulatory requirements. Baseline separation of the seven ethynyl steroids registered as oral contraceptives in the USA was obtained with the system 20 m M sodium borate-sodium phosphate buffer (pH 8) containing 50 m M sodium cholate and 10% (v/v) methanol. Factors that affect selectivity and reproducibility of the above separation systems are discussed.

Quantitative structure-chromatographic retention relationship study of six underivatized equine estrogens

Underivatized estrone (ES), equilin (EQ), equilenin (EQN) and their corresponding 17α-diols 17α-estradiol (ESD), 17α-dihydroequilin (DHEQ) and 17α-dihydroequilenin (DHEQN) were separated by TLC, RP-HPLC and capillary GC. Their dipole moments (μ) and Randić's connectivity indices ( 1 χ) were determined as parameters of importance for the separation. The number of H atoms was taken as an additive structural parameter of importance for the quantitative structure-chromatographic retention relationship study (QSRR). Principal component analysis (PCA) was applied in order to find similarities and dissimilarities between 9 TLC and 10 RP-HPLC systems. PCA indicated that proton donor-proton acceptor interactions play the most important role for the TLC and RP-HPLC separation. The two-dimensional non-linear map of PC variables showed that the keto-estrogens (ES, EQ and EQN) and the corresponding diols (ESD, DHEQ and DHEQN) form two separate clusters. The relationship between GC retention of equine estrogens characterized by Kováts indices (KI), their 1 χ and μ was expressed by the equation KI/100 = a/ 1 χ+ b/ μ 2+ c. The biological activity of the estrogens was related to log 1/μ 2.

Metabolism of equilin sulfate in the dog

The metabolism of equilin sulfate was determined in female dogs receiving 2.5 mg/kg of [ 3H]equilin sulfate alone or in a preparation that contained all the components that are present in the conjugated equine estrogen product Premarin ®. The pharmacokinetic parameters of total radioactivity indicated that the drug is rapidly absorbed and it has a moderate half-life in plasma. The total radioactivity in plasma following administration of [ 3H]equilin sulfate as part of a mixture of conjugated equine estrogens had significantly lower peak concentration ( C max), a lower area under the curve (AUC), a longer terminal half-life ( t 1 2 ) and a longer mean residence time (MRT) than when [ 3H]equilin sulfate was given alone, indicating that the other components in the conjugated equine estrogen preparation altered the pharmacokinetics of equilin sulfate. An average of 26.7 ± 4.4% of the administered radioactive dose was excreted in urine of dogs receiving [ 3H]equilin sulfate. Again, a significantly lower percentage (21.4 ± 6.3%, P = 0.023) was eliminated in urine of dogs receiving [ 3H]equilin sulfate in the conjugated equine estrogen preparation, indicating that the absorption of equilin sulfate was perhaps altered by the other components in the conjugated equine estrogen preparation. Metabolite profiles of plasma and urine were similar. Equilin, equilenin, 17β-dihydroequilenin, 17β-dihydroequilin, 17α-dihydroequilenin and 17α-dihydroequilin were present in both matrices. 17β-Dihydroequilin and equilin were the two major chromatographic peaks in plasma samples. 17β-Dihydroequilenin and 17β-dihydroequilin were the major metabolites in urine. In conclusion, following oral administration of [ 3H]equilin sulfate to dogs, the radioactivity is rapidly absorbed. The disposition of equilin sulfate is altered by the other components that are present in the conjugated equine estrogen preparation Premarin ®. The reduction of the 17-keto group and aromatization of ring-B are the major metabolic pathways of equilin in the dog.

Synthesis of [2,4-3H] 17β-dihydroequilin sulfate

[2,4-3H] 17β-dihydroequilin-3-sulfate ammonium salt suitable for in vivo pharmacokinetic studies was synthesized from [2,4-3H] equillin. Sulfation of [2,4-3H] equillin with pyridine-chiorosulfonic acid mixture gave in high yields [2,4-3H] equilin sulfate, which was then reduced with sodium borohydride to yield [2,4-3H] 17β-dihydroequilin sulfate. The reduction was sterospecific and no 17α-reduced products were formed.

Pharmacokinetics of 17 beta-dihydroequilin sulfate and 17 beta-dihydroequilin in normal postmenopausal women.

The MCRs of 17 beta-dihydroequilin sulfate and 17 beta-dihydroequilin were determined in normal postmenopausal women by single iv injection of either 17 beta-[3H]dihydroequilin sulfate ([3H]17 beta-EqS) or 17 beta-[3H]dihydroequilin ([3H]17 beta-Eq). After the administration of [3H]17 beta-EqS, blood was drawn at various time intervals, and the plasma obtained was fractionated into the unconjugated, sulfate, and glucuronide fractions. The bulk of radioactivity was present in the sulfate fraction, and from this [3H]17 beta-EqS, [3H]equilin sulfate, [3H]equilenin sulfate, and 17 beta-[3H]dihydroequilenin sulfate were isolated and purified, and their concentrations were measured. The disappearance of [3H]17 beta-EqS from plasma can be described as a function of two exponentials. The half-life of the initial fast component was 5 +/- 0.2 min; this component represents the distribution and transfer from a space, with a mean volume (V1) of 6 +/- 0.5 L. The value for the rate constant (k) of total removal from this space was 300 +/- 20 U/day, of which 35 +/- 2% was irreversible. The mean half-life of the slower component of 17 beta-EqS was 147 +/- 15 min, and the mean MCR was 376 +/- 93 L/day.m2. Similarly, after the administration of [3H]17 beta-Eq, the disappearance of radioactivity as 17 beta-Eq from plasma also had two components. The half-lives of the fast and slow component were 5.5 +/- 0.8 and 45 +/- 2.0 min, respectively. The MCR of 17 beta-Eq was 1252 +/- 103 L/day.m2. From both series of experiments, unconjugated and sulfate-conjugated equilin, equilenin, and 17 beta-dihydroequilenin were isolated and purified, and their concentrations were measured. No 17 alpha-reduced metabolites were detected. These results indicate that 17 beta-EqS is cleared twice as fast as equilin sulfate (MCR, 176 L/day.m2), whereas the more potent estrogen 17 beta-Eq is cleared 2 times slower than equilin. The slower elimination and greater estrogenic activity of 17 beta-Eq support the hypothesis that the major in vivo activity of equilin sulfate present in conjugated equine estrogen preparations is expressed via its metabolites 17 beta-EqS and 17 beta-Eq.

Metabolic clearance rate of equilin sulfate and its conversion to plasma equilin, conjugated and unconjugated equilenin, 17 beta-dihydroequilin, and 17 beta-dihydroequilenin in normal postmenopausal women and men under steady state conditions.

The constant infusion of [3H]equilin sulfate ([3H]EqS) was used to estimate the MCR of equilin sulfate (EqS) and to measure the conversion of this estrogen to equilin (Eq), equilenin (Eqn), equilenin sulfate (EqnS), 17 beta-dihydroequilin (17 beta-Eq), 17 beta-dihydroequilin sulfate (17 beta-EqS), 17 beta-dihydroequilenin (17 beta-Eqn), and 17 beta-dihydroequilenin sulfate (17 beta-EqnS) in normal postmenopausal women and men. Infusion of [3H]EqS was started in five postmenopausal women and two men 30 min after a priming dose and continued at a constant rate of 12-15 microCi/h for 3 h. Blood samples were taken 15 min before the end of infusion, at the end of the infusion, and 15 min after the end of infusion. Unconjugated and sulfate-conjugated Eq, Eqn, 17 beta-Eq, and 17 beta-Eqn were isolated from plasma. The mean MCR of EqS was calculated to be 280 +/- 24 L/day or 170 +/- 18 L/day.m2. The mean conversion ratios for precursor EqS to product 17 beta-EqS, EqnS, 17 beta-EqnS, 17 beta-Eq, Eq, Eqn, and 17 beta-Eqn were 0.300, 0.190, 0.100, 0.020, 0.016, 0.008, and 0.004 respectively. In both the sulfate-conjugated and unconjugated forms, 17 beta-Eq was the most abundant metabolite formed. 17 beta-Eq estrogen is a potent uterotropic agent and has a much higher affinity for estrogen receptors than Eq. Its formation may be of importance in the overall biological activity of EqS present in conjugated equine estrogen preparations.

Metabolic clearance rate of equilin sulfate and its conversion to plasma equilin, conjugated and unconjugated equilenin, 17 beta- dihydroequilin, and 17 beta-dihydroequilenin in normal postmenopausal women and men under steady state conditions

Metabolic clearance rate of equilin sulfate and its conversion to plasma equilin, conjugated and unconjugated equilenin, 17 beta- dihydroequilin, and 17 beta-dihydroequilenin in normal postmenopausal women and men under steady state conditions

Metabolism of [ 3H]equilin in normal and malignant human endometrium and in endometrial adenocarcinoma transplanted into nude mice

One of the main components of conjugated equine estrogens is equilin sulfate and this estrogen in postmenopausal women is metabolized to 17β-dihydroequilin, 17β-dihydroequilenin and equilenin. To investigate the possibility that some of these estrogens may be formed directly in the target tissues, we studied the in vitro metabolism of [ 3H]equilin in various types of normal and malignant human endometrium, including adenocarcinoma grown in athymic nude mice. The results indicate that normal and neoplastic human endometrium can form the above three metabolites. The highest level of 17β-reduced products were isolated from the normal secretory endometrium. Equilenin was the most abundant metabolite isolated from both the normal and malignant endometrium. The formation of [ 3H]equilenin indicates the presence of a 6,8(9) steroid dehydrogenase-isomerase in the human endometrium. The formation of 17β-dihydroequilin in the endometrium may be of importance as this estrogen is 8 times more potent as a uterotrophic agent than equilin and estrone.