替代对照品法测定新疆孕马尿中3种主要结合雌激素含量

目的 建立HPLC替代对照品法测定新疆孕马尿中3种主要结合雌激素的含量。方法 采用HPLC在不同条件下测定替代对照品雌酮硫酸钠对于马烯雌酮硫酸钠及17α-双氢马烯雌酮硫酸钠对照品的校正因子,利用校正因子和替代对照品雌酮硫酸钠测定孕马尿中3种主要结合雌激素的含量。结果 以替代对照品法测得的结果与常规含量测定方法结果一致。结论 在高效液相色谱仪上使用替代对照品法测定孕马尿中的3种主要结合雌激素的含量,此方法准确可靠,可节约检测成本。     

Conjugated estrogens bioinequivalence: comparison of four products in postmenopausal women.

The bioequivalence of four conjugated estrogens tablets USP was compared by measurement of seven estrogens or estrogen metabolites in the urine during steady-state dosing in postmenopausal women. Two studies compared three generic products with the innovator's product. The urinary excretion of 17 alpha-dihydroequilin, 17 alpha-dihydroequilenin, and 17 alpha-estradiol were significantly greater in all cases with the innovator's product than with the generic products. Statistically significant differences between products were observed occasionally for other components. The generic products thus were bioinequivalent to the innovator's product, although all products essentially met current compendial specifications. A third study observed no significant differences between three batches of the innovator's product for the seven components. Total conjugated estrogens excretion of all products at the steady state was essentially equal and correlated with neither disintegration time nor dissolution half-time. Bioinequivalence between products is discussed in relation to the need for an improved USP conjugated estrogens monograph. Evidence suggesting the metabolism of a fraction of dosed estrone, equilin, and 17 alpha-dihydroequilin to 17 beta-estradiol, 17 beta-dihydorequilim, and 17 alpha-dihydroequilenin, respectively, is presented.     

结合雌激素阴道软胶囊质量研究

目的建立结合雌激素阴道软胶囊（Conjugated Estrogens vaginal soft capsule）质量标准。方法采用高效液相色谱法测定结合雌激素阴道软胶囊含量;检查其崩解、融变时限,并与参比制剂进行对比研究。结果马烯雌酮硫酸钠和雌酮硫酸钠分别在2．02～10．09μg、4．03～20．16μg内呈良好的线性关系（r=0．9999）,马烯雌酮硫酸钠3个浓度回收率分别为100．0％,101．3％和99．4％,雌酮硫酸钠3个浓度回收率分别为99．0％,99．4％和101．8％。结合雌激素阴道软胶囊崩解、融变时限与参比制剂间无差异（P〉0．05）。结论本法专属性强,重复性、精密度好,结果准确可靠,可作为结合雌激素阴道软胶囊的质量控制方法。     

Single dose pharmacokinetics and bioequivalence of conjugated estrogens (0.625 mg × 2) tablets in healthy post-menopausal female subjects in fasting and fed studies

Abstract

Conjugated estrogens are sulfate esters of naturally occurring estrogens. The pharmacokinetics of various estrogen formulations is complex and varying due to its endogenous availability. The present studies were designed to evaluate pharmacokinetic parameters and bioequivalence between two formulations of conjugated estrogens (0.625?mg tablets). Both the studies were designed as two-treatment, four-period, replicate cross-over single dose studies in 60 healthy post-menopausal female subjects under fasting and fed conditions, respectively. Since estrone is present endogenously, for baseline correction three pre-dose samples were obtained for total and unconjugated estrone. Plasma samples were analyzed by validated LC-MS/MS method and pharmacokinetic parameters were estimated for total and unconjugated forms of both estrone and equilin. The least square mean ratios and its 90% confidence interval for primary pharmacokinetic parameters C_max, AUC_0–t and AUC_0–inf were found to be within bioequivalence limits of 80.00–125.00% for total and unconjugated forms of baseline corrected estrone and baseline un-corrected equilin. In conclusion, both test and reference products were well-tolerated and the test product was bioequivalent with the reference product in terms of the rate and extent of absorption in both fasting and fed studies.     

Densitometric determination of conjugated oestrogens in the raw material and in pharmaceutical preparations

A densitometric method for determination of complex mixtures of conjugated oestrogens in raw material and tablets was developed. The proposed procedure comprised hydrolysis of sodium sulphate esters of oestrone, equilin, 17 alpha-oestradiol, equilenin, 17 alpha-dihydroequilin and 17 alpha-dihydroequilenin, the chloroform extraction of free oestrogens and methyltestosterone (internal standard) and separation on TLC plates using chloroform-cyclohexane-dioxane-triethylamine (4.5:4.1:0.6, v/v) as the solvent for development. Quantitative assay was achieved by direct scanning of the oestrogen spots at 280 nm. The proposed method is simple, rapid, reproducible and adequate to control the content of conjugated oestrogens in the raw material and in pharmaceutical preparations.     

Evaluation of single- and multiple-dose pharmacokinetics of synthetic conjugated estrogens, A (Cenestin) tablets: a slow-release estrogen replacement product

A multiple-dose, placebo-controlled, randomized pharmacokinetic study was performed in 15 early (i.e., 1-3 years) postmenopausal women to evaluate the single and steady-state pharmacokinetics of 0.625 mg Cenestin (Synthetic Conjugated Estrogens, A) tablets, administered once daily for 90 days. Plasma concentration-time profiles for both total (conjugated and unconjugated) estrone and equilin, two major estrogens in Cenestin, were nearly superimposable between Day 1 (single dose) and Day 90 (multiple dose), indicating equivalent drug exposure from one dose to the next. For total estrone, the mean estimates of Cmax and AUC0-24 were 2.5 ng/ml and 35.0 ng x h/ml for Day 1 and 3.0 ng/ml and 39.8 ng x h/ml for Day 90, respectively. Similarly, Cmax and AUC0-24 mean values for total equilin were 1.4 ng/ml and 17.4 ng x h/ml after Day 1 and 1.5 ng/ml and 17.3 ng x h/ml after Day 90, respectively. The mean tmax value for total estrone was 8.3 hours on Day 1 and 8.6 hours on Day 90, indicating a slower rate of absorption. The average estimate for observed drug accumulation index for the 24-hour dosing interval was calculated to be 1.1 for total estrone and 1.0 for total equilin. These data, taken together, indicate a slow and steady rate of absorption, minimal drug accumulation at steady state, and consistent drug exposure between Cenestin doses.     

Estrogens: their function, uses and hazards. Part 1

After explaining the role of estrogens in normal human physiology with a discussion of fertilization which mentions cytogenetical research, the history of estrogen synthesis, forms of estrogen, and the roles estrogens play in female physiology after birth are discussed. The observation that estrus in an animal was mediated by a chemical agent was made in 1922. In 1927, studies showed that urine of pregnant women contained estrogenic substances, and in 1929, estrone, an oxidation product of estradiol, was isolated from this source. The natural oxidation products of estradiol include, besides estrone, estriol, equilin, and equilenin, these last 3 being very weak estrogens. Conjugated estrogens consist chiefly of sodium estrone sulfate. In 1938, diethylstilbestrol, a nonsteroidal estrogen, was synthesized. A discussion of the estrogens activities when administered orally or parenterally follows; nonsteroidal estrogens are thought to have good oral action as do esterified estrogens. Estrogenic effects on female physiology include secondary sexual characteristics, menstrual cycle, and ovulation. Other estrogen functions mentioned were antidiuretic action, retardation of atherosclerosis, increased calcium deposition in bone, and epiphyseal closure acceleration after initial stimulation. The rationale for and problems with patient package inserts are discussed.     

Separation and quantitation of esterified estrogens in bulk mixtures and combination drug preparations using high-performance liquid chromatography.

A high-performance liquid chromatographic method for esterified estrogens is described. By using a facile acid hydrolysis extraction procedure for the sample preparation, the compounds are chromatographed as their free phenolic forms. The separation of structurally similar compounds, such as equilenin, equilin, estrone, and estradiol, was achieved with a reversed-phase column and a methanol--water mobile phase. Several samples of bulk mixtures and tablets were assayed; the results compared favorably with those obtained using the USP XIX method. The method was rapid, and the detector response was linear over a wide concentration range. A relative standard deviation of +/- 5% indicates the reliability and accuracy of the proposed method.     

Liquid chromatographic analysis of conjugated and esterified estrogens in tablets

A high-performance liquid chromatographic (HPLC) method was developed for the analysis of conjugated and esterified estrogens in tablets. The 3-sulfate estrogen derivatives were hydrolyzed with acid, mixed with ethinyl estradiol (the internal standard), and extracted into benzene. The liberated 3-phenolic estrogens were converted to dansyl derivatives, separated on an adsorption column, and detected fluorometrically. Estrogens with a 17-keto functional group required a prior reduction to the corresponding 17 beta-hydroxyl derivatives with sodium borohydride in order to be chromatographically resolved. The validity of the proposed method was demonstrated by analyzing samples of commercial tablets of conjugated and esterified estrogens of various strengths.     

Determination of aqueous solubility and pKa values of estrogens.

Reported estrone pKa and solubility data show wide variation. Improved experimental procedures were designed and used to obtain reproducible results. The pKa values for several estrogens and related compounds also were determined to assess the effects of structural differences on ionization. No evidence was obtained for long-range D to A ring electronic transmission affecting pKa. Significant differences in pKa values resulted only when conjugated unsaturation was added into the B ring of estrone or estradiol. The aqueous solubilities of estrone and 17alpha-estradiol were 0.8 and 3.9 microgram/ml, respectively, at 25degrees.     

新疆孕马尿中主要结合雌激素SPE-AgNO_3-TLC定性鉴别研究

目的:建立孕马尿中主要结合雌激素的SPE-AgNO3-TLC定性方法。方法:采用大孔树脂固相萃取柱对孕马尿中结合雌激素富集净化,通过盐洗、碱洗除去部分杂质,乙醇进行洗脱,采用AgNO3-TLC鉴别孕马尿中3种主要结合雌激素。结果:薄层色谱中,供试品色谱与对照品色谱在相应位置显相同颜色的条带,条带清晰,分离效果好,阴性无干扰。结论:SPE-AgNO3-TLC适合孕马尿中主要结合雌激素的定性检测,对于孕马尿中结合雌激素品质评价具有重要参考价值。     

Determination of estrogens in dosage forms by fluorescence using dansyl chloride.

A sensitive, reproducible, fluorometric procedure for the determination of estrogens in pharmaceutical preparations is presented. The estrogens are determined fluorometrically following their reaction with dansyl chloride. The optimum conditions for the reaction such as pH, reaction solvent composition variations, and speed of reaction are discussed. In addition, a linearity study of the relationship between concentration and fluorescence intensity for estrone, estradiol, and ethinyl estradiol is reported. Solvent extraction procedures based on acid-base behavior or column chromatography are used when necessary to isolate the estrogen prior to reaction with dansyl chloride and fluorometric measurement. The recovery of estrogens from spiked samples indicated that the proposed method is efficient and reproducible. Comparison of the dansyl procedure with the official NF method in the analysis of estrone aqueous suspension showed the proposed method to be accurate and more precise than the NF assay. Estrone aqueous suspension, estradiol in sesame oil, estradiol valerate in castor oil and in sesame oil (in the latter case, in the presence of testosterone), estradiol benzoate in sesame oil, and ethinyl estradiol tablets (0.5 mg/tablet) from commercial sources were satisfactorily analyzed, with average results within compendial limits and no coefficient of variation greater than 2%.     

The major metabolite of equilin, 4-hydroxyequilin, autoxidizes to an o-quinone which isomerizes to the potent cytotoxin 4-hydroxyequilenin-o-quinone

The risk factors for women developing breast and endometrial cancers are all associated with a lifetime of estrogen exposure. Estrogen replacement therapy in particular has been correlated with a slight increased cancer risk. Previously, we showed that equilenin, a minor component of Premarin (Wyeth-Ayerst), was metabolized to highly cytotoxic quinoids which caused oxidative stress and alkylation of DNA in vitro [Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. (1998) Chem. Res. Toxicol. 11, 1113-1127]. In this study, we have compared the chemistry of the major catechol metabolite of equilin (4-hydroxyequilin), which is found in several estrogen replacement formulations, to the equilenin catechol (4-hydroxyequilenin). Unlike endogenous catechol estrogens, both equilin and equilenin were primarily converted by rat liver microsomes to 4-hydroxylated rather than 2-hydroxylated o-quinone GSH conjugates. With equilin, a small amount of 2-hydroxyequilin GSH quinoids were detected (4-hydroxyequilin:2-hydroxyequilin ratio of 6:1); however, no peaks corresponding to 2-hydroxyequilenin were observed in incubations with equilenin. These data suggest that unsaturation in the B ring alters the regiochemistry of P450-catalyzed hydroxylation from primarily 2-hydroxylation for endogenous estrogens to 4-hydroxylation for equine estrogens. 4-Hydroxyequilenin-o-quinone reacts with GSH to give two mono-GSH conjugates and one di-adduct. The behavior of 4-hydroxyequilin was found to be more complex than 4-hydroxyequilenin as conjugates resulting from 4-hydroxyequilenin were detected in addition to the 4-hydroxyequilin-GSH adducts. The mechanism of decomposition of 4-hydroxyequilin likely involves isomerization to a quinone methide which readily aromatizes to 4-hydroxyequilenin followed by autoxidation to 4-hydroxyequilenin-o-quinone. Similar results were obtained with 2-hydroxyequilin, although, in contrast to 4-hydroxyequilenin, 2-hydroxyequilenin does not autoxidize and the reaction stops at the catechol. Since 4-hydroxyequilin is converted to 4-hydroxyequilenin and 4-hydroxyequilenin-o-quinone, similar effects were observed for this equine catechol, including consumption of NAD(P)H likely by the 4-hydroxyequilenin-o-quinone, depletion of molecular oxygen by 4-hydroxyequilenin or its semiquinone radical, and alkylation of deoxynucleosides and DNA by 4-hydroxyequilenin quinoids. Finally, preliminary studies conducted with the human breast tumor cell line MCF-7 demonstrated that the cytotoxic effects of the catechol estrogens from estrone, equilin, and 2-hydroxyequilenin were similar, whereas 4-hydroxyequilenin was a much more potent cytotoxin ( approximately 30-fold). These results suggest that the catechol metabolites of equine estrogens have the ability to cause alkylation/redox damage in vivo primarily through formation of 4-hydroxyequilenin quinoids.     

Simultaneous quantification of four native estrogen hormones at trace levels in human cerebrospinal fluid using liquid chromatography-tandem mass spectrometry

Estrogens are known to exhibit neuroprotective effects on the brain. Their importance in this regard and in others has been emphasized in many recent studies, which increases the need to develop reliable analytical methods for the measurement of estrogen hormones. A heart-cutting two-dimensional liquid chromatography separation method coupled with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) has been developed for simultaneous measurement of four estrogens, including estriol (E3), estrone (E1), 17β-estradiol (17β-E2), and 17α-estradiol (17α-E2), in human cerebrospinal fluid (CSF). The method was based on liquid-liquid extraction and derivatization of estrogens with dansyl chloride to enhance the sensitivity of ESI-based detection in conjunction with tandem mass spectrometry. Dansylated estriol and estrone were separated in the first dimension by an amide-C18 column, while dansylated 17β- and 17α-estradiol were resolved on the second dimension by two C18 columns (175 mm total length) connected in series. This is the first report of a method for simultaneous quantification of all four endogenous estrogen compounds in their dansylated form. The detection limits for E1, 17α-E2, 17β-E2, and E3 were 19, 35, 26, and 61pg/mL, respectively. Due to matrix effects, validation and calibration was carried out in charcoal-stripped CSF. The precision and accuracy were more than 86% for the two E2 compounds and 79% for E1 and E3 while the extraction recovery ranged from 91% to 104%. The method was applied to measure estrogens obtained in a clinical setting, from the CSF of ischemic trauma patients. While 17β-estradiol was present at a significant level in the CSF of some samples, other estrogens were present at lower levels or were undetectable.     

Steady-state urinary excretion method for determining bioequivalence of conjugated estrogen products.

The steady-state excretion of conjugated estrogens in the urine of postmenopausal women dosed with conjugated estrogens tablets was studied using a modification of a previously published method. The procedure was used to quantitate the estrogens both before and during conjugated estrogens replacement therapy. The method, which is relatively specific, involves enzyme hydrolysis of urine samples a number of classical extraction and purification steps, and analysis of the silylated estrogens on a 2.7-m, 1.7% diethylene glycol succinate column using flame-ionization detection. The results indicate that steady-state urinary estrogen excretion levels were obtained within 17 days of dosing. Furthermore, the urinary estrogen excretion profile was significantly different from the composition of the estrogens in the dosage form.     

Breast cancer resistance protein exports sulfated estrogens but not free estrogens

Breast cancer resistance protein (BCRP), an ATP-binding cassette transporter, confers resistance to a series of anticancer reagents such as mitoxantrone, 7-ethyl-10-hydroxycamptothecin, and topotecan. We reported previously that estrone and 17beta-estradiol reverse BCRP-mediated multidrug resistance. In the present study, we demonstrate that BCRP exports estrogen metabolites. First, we generated BCRP-transduced LLC-PK1 (LLC/BCRP) cells, in which exogenous BCRP is expressed in the apical membrane, and investigated transcellular transport of 3H-labeled compounds using cells plated on microporous filter membranes. The basal-to-apical transport (excretion) of mitoxantrone, estrone, and 17beta-estradiol was greater in LLC/BCRP cells than in LLC-PK1 cells. Thin-layer chromatography of transported steroids revealed that the transport of estrone and 17beta-estradiol was independent of BCRP expression. Alternatively, increased excretion of estrone sulfate and 17beta-estradiol sulfate was observed in LLC/BCRP cells. BCRP inhibitors completely inhibited the increased excretion of sulfated estrogens across the apical membrane. Conversion of estrogens into their sulfate conjugates was similar between LLC/BCRP and LLC-PK1 cells, suggesting that the increased excretion of estrogen sulfates was attributable to BCRP-mediated transport. Next, the uptake of 3H-labeled compounds in membrane vesicles from BCRP-transduced K562 (K562/BCRP) cells was investigated. 3H-labeled estrone sulfate, but not 3H-labeled estrone or 17beta-estradiol, was taken up by membrane vesicles from K562/BCRP cells, and this was ATP-dependent. Additionally, BCRP inhibitors suppressed the transport of estrone sulfate in membrane vesicles from K562/BCRP cells. These results suggest that BCRP does not transport either free estrone or 17beta-estradiol but exports sulfate conjugates of these estrogens.     

HPLC analysis of pharmaceutical estrogens in raw materials and dosage forms

The use of HPLC with fluorescence detection (λ_ex=280 nm; λ_em=410 or 312 nm) in combination with a postcolumn on line photochemical derivatization was investigated for the analysis of conjugated and unconjugated estrogens and their correlated impurities. The column effluents were subjected on-line to UV irradiation (254 nm) and the photo induced modifications were useful for the identification of the various estrogens. The proposed HPLC methods were successfully applied to the analysis of commercially available conjugated estrogens (raw materials and pharmaceuticals) and estrogen samples. The assay results relative to the pharmaceutical formulations were in agreement with those obtained by a reference HPLC method with UV detection (λ=280 nm).     

CYTOTOXICITY OF ESTRADIOL,EQUILIN, EQUILENIN,AND THEIR DERIVATIVES ON CHINESE HAMSTER V79 CELLS

Recently, we investigated the inhibitory effects of 17β-estradiol and diethylstilbestrol on microtubule assembly, cytotoxicity, and aneuploidy in V79 cells. The present study analyzes the effects of equilin and equilenin (amongst the natural estrogens originally isolated from the urine of pregnant horses) and their related compounds, on the relative plating efficiency of Chinese hamster V79 cells. The results showed that a hydroxyl group on 17-C and a methoxyl group on 3-C of the estrogen skeleton were important for cytotoxicity. Of the various compounds analyzed, 2-methoxyestradiol had the strongest cytotoxicity, suggesting also the importance of a methoxyl group on 2-C.     

Isolation and identification of metabolites of 14C-labeled estradiol in cattle.

A total of six steers and six heifers received three daily injections containing either 200 muCi (1 mg) of [4-14C] estradiol-17beta or 312 muCi (2.16 mg) of [4-14C] estradiol 17beta 3-benzoate. Major metabolites of the administered estradiol-17beta and estradiol-17beta 3-benzoate were identified in muscle, fat, liver, and kidney samples obtained 3 hr after the final injection. Estradiol benzoate was nondetectable in the tissues analyzed, suggesting rapid hydrolysis of the benzoate ester. Consequently, the relative proportions of the various metabolites were similar for both the injected estrogens. Estradiol-17beta and estrone, which together accounted for 80-90% of the total extracted radioactivity, appear to be the major metabolites in both muscle and fats. In contrast, the major metabolites present in liver and kidney appear in the conjugate fraction. Most of the conjugated metabolites were glucuronates, which represent 85-95% of the total recovered conjugate radioactivity.