Direct quantification of anorethidrani disuccinate and determination of sterol metabolites by chemical derivatization combined with LC-MS/MS: Application to a Phase I pharmacokinetic study in humans.

Anorethidrani disuccinate (ACP) is a domestically designed A-decarbonized steroid that is currently being investigated in Phase I clinical trials for the treatment of solid tumors. Only the parent drug exhibited antitumor activity; its sterol metabolite M2 showed obvious antiestrogenic effects. We have developed a rapid, sensitive, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the direct quantification of ACP and a chemical derivatization method that can be used to quantify M2 derivatized with glycidyl trimethyl ammonium chloride (GTMA). A simple protein precipitation procedure was performed to quantify ACP. Injections were obtained within 3.5 min on an Eclipse Plus Phenyl-Hexyl column (50 mm × 2.1 mm i.d., 1.8 µm) with gradient elution; the calibration curve was linear over the range of 2.00-8000 ng/mL. For quantification of M2 in plasma, analytes were extracted by protein precipitation and converted to their GTMA derivatives at 60 °C for 2 h at pH 12; the analytes and coelutants were separated on a Luna C8(2) column (50 mm × 2.0 mm i.d., 5.0 µm). The precision (RSD) and accuracy (RE) of the intra- and interday determinations were within 10%. The derivatization procedure is a novel method for sterol determination by LC-MS/MS. The results confirmed the usefulness of this method for characterizing the pharmacokinetic profiles of ACP and its major metabolite M2 in a Phase I pharmacokinetic study.

The synthesis of 4-chloro-17ß-hydroxymethyl-17α-methyl-18-norandrosta-4,13-diene-3α-ol - Proposed long term metabolite (M4) of oralturinabol.

4-Chloro-17ß-hydroxymethyl-17α-methyl-18-norandrosta-4,13-diene-3α-ol is one of proposed long term metabolites of oralturinabol (anabolic androgenic steroid restricted in sport). The synthesis of 4-chloro-17ß-hydroxymethyl-17α-methyl-18-norandrosta-4,13-diene-3α-ol was achieved. Isomerisation of configuration of 13-carbon was used for construction of 17ß-hydroxymethyl-17α-methyl fragment. The proposed route of synthesis allows to obtain 3ß-hydroxy isomer as well.

Predominant contributions of carboxylesterase 1 and 2 in hydrolysis of anordrin in humans.

1. Anordrin (2α, 17α-diethynyl-A-nor-5α-androstane-2ß, 17ß-diol diproprionate) is post-coital contraceptive drug that is on the market in China for more than 30 years. This study aims to elucidate enzymes involved in anordrin hydrolysis, and to evaluate the significant role of carboxylesterases in anordrin hydrolysis in humans. 2. Human liver and intestinal microsomes, recombinant human carboxylesterase were selected as enzyme sources. In human liver microsomes, intrinsic clearance was 684 ± 83 µL/min/mg protein, which was considerably higher than the value of intestine microsomes (94.6 ± 13.3 µL/min/mg protein). Carboxylesterase (CES) 1 has more contribution than CES2 in human liver. 3. Inhibition studies were performed using representative esterase inhibitors to confirm esterase isoforms involved in anordrin hydrolysis. Simvastatin strongly inhibited hydrolytic process of anordrin in liver and intestine microsomes, with IC50 values of 10.9 ± 0.1 and 6.94 ± 0.03 µM, respectively. 4. The present study investigated for the first time hydrolytic enzyme phenotypes of anordrin. Anordrin is predominantly catalyzed by CES1 and CES2 to generate the main active metabolite, anordiol. Moreover, anordrin and its metabolite anordiol can be altered by esterase inhibitors, such as simvastatin, upon exposure in vivo.

Anordrin Eliminates Tamoxifen Side Effects without Changing Its Antitumor Activity.

Tamoxifen is administered for estrogen receptor positive (ER+) breast cancers, but it can induce uterine endometrial cancer and non-alcoholic fatty liver disease (NAFLD). Importantly, ten years of tamoxifen treatment has greater protective effect against ER+ breast cancer than five years of such treatment. Tamoxifen was also approved by the FDA as a chemopreventive agent for those deemed at high risk for the development of breast cancer. The side effects are of substantial concern because of these extended methods of tamoxifen administration. In this study, we found that anordrin, marketed as an antifertility medicine in China, inhibited tamoxifen-induced endometrial epithelial cell mitosis and NAFLD in mouse uterus and liver as an anti-estrogenic and estrogenic agent, respectively. Additionally, compared with tamoxifen, anordiol, the active metabolite of anordrin, weakly bound to the ligand binding domain of ER-α. Anordrin did not regulate the classic estrogen nuclear pathway; thus, it did not affect the anti-tumor activity of tamoxifen in nude mice. Taken together, these data suggested that anordrin could eliminate the side effects of tamoxifen without affecting its anti-tumor activity.

C-H Acetoxylation-Based Chemical Synthesis of 17 ß-Hydroxymethyl-17 α-methyl-18-norandrost-13-ene Steroids.

Palladium-catalyzed C-H acetoxylation has been proposed as a key transformation in the first chemical synthesis of steroids bearing a unique 17ß-hydroxymethyl-17α-methyl-18-nor-13-ene D-fragment. This C-H functionalization step was crucial for inverting the configuration at the quaternary stereocenter of a readily available synthetic intermediate. The developed approach was applied to prepare the metandienone metabolite needed as a reference substance in anti-doping analysis to control the abuse of this androgenic anabolic steroid.

Synthesis of novel 13α-18-norandrostane-ferrocene conjugates via homogeneous catalytic methods and their investigation on TRPV1 receptor activation.

13α-Steroid-ferrocene derivatives were synthesized via two reaction pathways starting from an unnatural 16-keto-18-nor-13α-steroid. The unnatural steroid was converted to ferrocene derivatives via copper-catalyzed azide-alkyne cycloaddition or palladium-catalyzed aminocarbonylation. 16-Azido- and 16-N-(prop-2-ynyl)-carboxamido-steroids were synthesized as starting materials for azide-alkyne cycloaddition with the appropriate ferrocene derivatives. Based on our earlier work, aminocarbonylation of 16-iodo-16-ene and 16-iodo-15-ene derivatives was studied with ferrocenylmethylamine. The new products were obtained in moderate to good yields and were characterized by (1)H and (13)C NMR, IR and MS. The solid state structure of the starting material 13α-18-norandrostan-16-one and two carboxamide products were determined by X-ray crystallography. Evidences were provided that the N-propargyl-carboxamide compound as well as its ferrocenylmethyltriazole derivative are able to decrease the activation of TRPV1 receptor on TRG neurons.

Biosynthesis of a steroid metabolite by an engineered Rhodococcus erythropolis strain expressing a mutant cytochrome P450 BM3 enzyme.

In the present study, the use of Rhodococcus erythropolis mutant strain RG9 expressing the cytochrome P450 BM3 mutant M02 enzyme has been evaluated for whole-cell biotransformation of a 17-ketosteroid, norandrostenedione, as a model substrate. Purified P450 BM3 mutant M02 enzyme hydroxylated the steroid with >95 % regioselectivity to form 16-ß-OH norandrostenedione, as confirmed by NMR analysis. Whole cells of R. erythropolis RG9 expressing P450 BM3 M02 enzyme also converted norandrostenedione into the 16-ß-hydroxylated product, resulting in the formation of about 0.35 g/L. Whole cells of strain RG9 itself did not convert norandrostenedione, indicating that metabolite formation is P450 BM3 M02 enzyme mediated. This study shows that R. erythropolis is a novel and interesting host for the heterologous expression of highly selective and active P450 BM3 M02 enzyme variants able to perform steroid bioconversions.

Identification of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid and ß-oxidation products of the C-17 side chain in cholic acid degradation by Comamonas testosteroni TA441.

Comamonas testosteroni degrades testosterone into 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and 2-hydroxyhexa-2,4-dienoic acid via aromatization of the A-ring. The former compound is suggested to be degraded further by ß-oxidation, but the details of the process remain unclear. In this study, we identified 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid as an intermediate compound in the ß-oxidation of this compound. ORF32, located in one of the two main steroid degradation gene clusters, was shown to be indispensable for the conversion of this compound. A homology search indicated that ORF32 encodes a hydratase for the CoA-ester, suggesting that ORF32 encodes a hydratase that adds a water molecule to a double bond at C-6 of the CoA-ester of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid. From the culture of an ORF32-disrupted mutant incubated with cholic acid for a short period (around two days, when a considerable number of intermediate compounds were detected by HPLC), 7α,12α-dihydroxy-3-oxochola-1,4-dien-24-oic acid, 7α,12α-dihydroxy-3-oxochol-4-en-24-oic acid, 12α-hydroxy-3-oxochola-4,6-dien-24-oic acid, 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylic acid, 12α-hydroxy-3-oxopregna-4,6-diene-20-carboxylic acid, 7α,12α-dihydroxy-3-oxopregn-4-ene-20-carboxylic acid, 12α-hydroxy-3-oxopregna-4,6-diene-20-carboxylic acid, 7α-hydroxy-3-oxopregna-4,17(20)-diene-20-carboxylic acid, and 3-oxopregna-4,6,17(20)-triene-20-carboxylic acid were isolated as intermediate compounds of C-17 side-chain degradation. The presence of these compounds implies that the process of degradation of the C-17 side chain in C. testosteroni will be similar to the process in Pseudomonas. The final two compounds, which have a double bond at the C-17(20) position, are here identified for the first time, to the best of our knowledge, as intermediate compounds in bacterial steroid degradation; their composition suggests that the remaining three carbons at the C-17 position would be removed oxidatively as a propionic acid derivative.

miR-331-3p regulates expression of neuropilin-2 in glioblastoma.

Aberrant expression of microRNAs (miRNAs), a class of small non-coding regulatory RNAs, has been implicated in the development and progression of high-grade gliomas. However, the precise mechanistic role of many miRNAs in this disease remains unclear. Here, we investigate the functional role of miR-331-3p in glioblastoma multiforme (GBM). We found that miR-331-3p expression in GBM cell lines is significantly lower than in normal brain, and that transient overexpression of miR-331-3p inhibits GBM cell line proliferation and clonogenic growth, suggesting a possible tumor suppressor role for miR-331-3p in this system. Bioinformatics analysis identified neuropilin-2 (NRP-2) as a putative target of miR-331-3p. Using transfection studies, we validated NRP-2 mRNA as a target of miR-331-3p in GBM cell lines, and show that NRP-2 expression is regulated by miR-331-3p. RNA interference (RNAi) to inhibit NRP-2 expression in vitro decreased the growth and clonogenic growth of GBM cell lines, providing further support for an oncogenic role for NRP-2 in high-grade gliomas. We also show that miR-331-3p inhibits GBM cell migration, an effect due in part to reduced NRP-2 expression. Finally, we identified a significant inverse correlation between miR-331-3p and NRP-2 expression in The Cancer Genome Atlas GBM cohort of 491 patients. Together, our results suggest that a loss of miR-331-3p expression contributes to GBM development and progression, at least in part via upregulating NRP-2 expression and increasing cell proliferation and clonogenic growth.

A synthetic 18-norsteroid distinguishes between two neuroactive steroid binding sites on GABAA receptors.

In the absence of GABA, neuroactive steroids that enhance GABA-mediated currents modulate binding of [35S]t-butylbicyclophosphorothionate in a biphasic manner, with enhancement of binding at low concentrations (site NS1) and inhibition at higher concentrations (site NS2). In the current study, compound (3alpha,5beta,17beta)-3-hydroxy-18-norandrostane-17-carbonitrile (3alpha5beta-18-norACN), an 18-norsteroid, is shown to be a full agonist at site NS1 and a weak partial agonist at site NS2 in both rat brain membranes and heterologously expressed GABAA receptors. 3alpha5beta-18-norACN also inhibits the action of a full neurosteroid agonist, (3alpha,5alpha,17beta)-3-hydroxy-17-carbonitrile (3alpha5alphaACN), at site NS2. Structure-activity studies demonstrate that absence of the C18 methyl group and the 5beta-reduced configuration both contribute to the weak agonist effect at the NS2 site. Electrophysiological studies using heterologously expressed GABAA receptors show that 3alpha5beta-18-norACN potently and efficaciously potentiates the GABA currents elicited by low concentrations of GABA but that it has low efficacy as a direct activator of GABAA receptors. 3alpha5beta-18-norACN also inhibits direct activation of GABAA receptors by 3alpha5alphaACN. 3alpha5beta-18-norACN also produces loss of righting reflex in tadpoles and mice, indicating that action at NS1 is sufficient to mediate the sedative effects of neurosteroids. These data provide insight into the pharmacophore required for neurosteroid efficacy at the NS2 site and may prove useful in the development of selective agonists and antagonists for neurosteroid sites on the GABAA receptor.

Hydrogen bonding between the 17beta-substituent of a neurosteroid and the GABA(A) receptor is not obligatory for channel potentiation.

BACKGROUND AND PURPOSE: Potentiating neurosteroids are some of the most efficacious modulators of the mammalian GABA(A) receptor. One of the crucial interactions may be between the C20 ketone group (D-ring substituent at C17) of the neurosteroid, and the N407 and Y410 residues in the M4 domain of the receptor. In this study, we examined the contribution of hydrogen bonding between 17beta-substituents on the steroid D-ring and the GABA(A) receptor to potentiation by neurosteroids. EXPERIMENTAL APPROACH: Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing wild-type and mutant alpha1beta2gamma2L GABA(A) receptors. KEY RESULTS: A steroid with a 17beta-carbonitrile group (3alpha5alpha18nor17betaCN) was a potent and efficacious potentiator of the GABA(A) receptor. Potentiation was also shown by a cyclosteroid in which C21 and the C18 methyl group of (3alpha,5alpha)-3-hydroxypregnan-20-one are connected within a six-membered ring containing a double bond as a hydrogen bond acceptor (3alpha5alphaCDNC12), a steroid containing a 17beta-ethyl group on the D-ring (3alpha5alpha17betaEt) and a steroid lacking a 17beta-substituent on the D-ring (3alpha5alpha17H). Single-channel kinetic analysis indicates that the kinetic mechanism of action is the same for the neurosteroid 3alpha5alphaP, 3alpha5alpha18nor17betaCN, 3alpha5alphaCDNC12, 3alpha5alpha17betaEt and 3alpha5alpha17H. Interestingly, 3alpha5alpha17betaEt, at up to 3 microM, was incapable of potentiating the alpha1N407A/Y410F double mutant receptor. CONCLUSIONS AND IMPLICATIONS: Hydrogen bonding between the steroid 17beta-substituent and the GABA(A) receptor is not a critical requirement for channel potentiation. The alpha1N407/Y410 residues are important for neurosteroid potentiation for reasons other than hydrogen bonding between steroid and receptor.

ORF18-disrupted mutant of Comamonas testosteroni TA441 accumulates significant amounts of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and its derivatives after incubation with steroids.

In a steroid degradation gene cluster of Comamonas testosteroni TA441 consisting of ORF18, 17 and tesIHA2A1DEFG, ORF18 was implicated in encoding a CoA-transferase by database searches, but the matching substrate was not clear. In this study, ORF18 was shown to be necessary for conversion of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, a product of hydrolysis of 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid in steroid degradation by TA441. The ORF18-disrupted mutant accumulates 7-hydroxy-9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and 7,12-dihydroxy-9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid when incubated with chenodeoxycholic acid and cholic acid, respectively.

Neuroactive steroids have multiple actions to potentiate GABAA receptors.

The effects of neuroactive steroids on the function of GABAA receptors were studied using cell-attached records of single channel activity recorded from HEK293 cells transfected with alpha1 beta2 gamma2L subunits. Activity was elicited with a half-maximal (50 microM) concentration of GABA. Two steroids were studied in detail: ACN ((3alpha,5alpha,17beta)-3-hydroxyandrostane-17-carbonitrile) and B285 ((3alpha,5beta,17beta)-3-hydroxy-18-norandrostane-17-carbonitrile). Four effects on channel activity were seen, two on open time distributions and two on closed times. When clusters of openings were elicited in the absence of steroid, the open time distribution contained three components. ACN produced concentration-dependent alterations in the open time distribution. The prevalence of the longest duration class of open times was increased from about 15% to about 40% (EC50 about 180 nM ACN), while the duration of the longest class increased from 7.4 ms to 27 ms (EC50 about 35 nM ACN). B285 also increased the prevalence of the longest duration open times (EC50 about 18 nM B285) but increased the duration only at concentrations close to 10 microM. The differences in the actions of these two steroids suggest that the effects on proportion and duration of the long duration open time component are produced by independent mechanisms and that there are separate recognition sites for the steroids which are associated with the two functional actions. The closed time distributions also showed three components in the absence of steroid. The rate of occurrence of the two brief duration closed time components decreased with increasing ACN, with an EC50 of about 50 nM ACN. In contrast, B285 did not reduce the rate of occurrence of the brief closings until high concentrations were applied. However, both B285 and ACN reduced the rate of occurrence of the activation-related closed state selectively, with comparable IC50 concentrations (about 40 nM ACN, 20 nM B285). As in the case for action on open times these data suggest that there are two recognition sites and two independent mechanisms, perhaps the sites and mechanisms associated with actions on open times. The presence of 1 microM ACN had no effect on the estimated channel opening rate or on the apparent affinity of the receptor for GABA. Mutation of the carboxy terminus of the gamma2 subunit, but not the alpha1 or beta2 subunits, abolished the ability of ACN to increase the duration of OT3 but had no effect on the reduction of the rate of occurrence of the activation-related closed state. These observations are also consistent with the idea that there is more than one distinguishable steroid recognition site on the GABAA receptor.

16-Cyano-13alpha-(5-methyl-1,3,4-oxadiazol-2-yl)-13,16-seco-17-norandrost-5-en-3beta-yl acetate.

In the title compound, C(23)H(31)N(3)O(3), the outer cyclohexane rings have chair conformations, while the central cyclohexene ring adopts a half-chair conformation. In the solid state, intra- and intermolecular C-H.N interactions are observed.

Effects of acetazolamide and anordiol on osmotic water permeability in AQP1-cRNA injected Xenopus oocyte.

AIM: To study the effects of acetazolamide and anordiol on osmotic water permeability in aquaporin 1 (AQP1)-cRNA injected Xenopus oocyte and their mechanisms. METHODS: AQP1 gene constructed in pBluescript was transcripted into cRNA in vitro and then the cRNA was injected in Xenopus oocytes. The effects of acetazolamide and anordiol on the water transport function of AQP1 were observed by assaying the osmotic swelling of oocytes. In addition, their effects on protein expression of AQP1 were quantitatively investigated by Western blotting method. RESULTS: After incubation for 15 min or 72 h, acetazolamide, a carbonic anhydrase inhibitor, equally reduced the water permeability of AQP1-cRNA injected oocyte in a dose-dependent manner. After incubation for 72 h, anordiol, an antiestrogen with partial estrogenic activity, reduced the osmotic water permeability dose dependently as well; however, no discernable action was observed after incubation with anordiol for 15 min. The Western blotting analysis showed that acetazolamide did not influence the protein expression of AQP1. However, after incubation for 72 h with anordiol (10 micromol/L), the quantity of AQP1 in the oocyte membrane was decreased dramatically (P<0.05). CONCLUSION: Both acetazolamide and anordiol inhibited the osmotic water permeability of AQP1-cRNA injected oocyte, but their mechanisms were different. Acetazolamide functionally inhibited the osmotic water permeability of AQP1, whereas anordiol primarily decreased the amount of AQP1 protein in the oocyte membrane.

Anordiol produces pregnancy loss in the rat--via the embryo or via the uterus?

Anordiol, the dihydroxylated metabolite of anordrin, is an antiestrogen with estrogenic activity that is known to inhibit fertility. The following study was conducted to determine the mechanism of this antifertility effect. Anordiol was administered orally to rats, prior to implantation, on Day 2 of pregnancy. Control animals were treated with the vehicle only. The effectiveness of the agent in terminating pregnancy was determined on Day 14 of pregnancy. Anordiol was 100% effective in abolishing pregnancy at a dose of 0.6 mg/Kg. Administration of smaller doses resulted in a decreased number of implanting embryos, in a dose-dependent manner. An additional dose of anordiol on Day 3 of pregnancy yielded similar results. To determine whether pregnancy impairment by anordiol is exerted via the embryo or via the uterus, reciprocal embryo transfers were performed. Day 5 blastocysts were transferred into the uteri of pseudopregnant rats. In one set of experiments, the donor rats were treated with anordiol, and in the second set the recipient rats were treated. The results indicate that the effects of anordiol administration are exerted via the embryo as well as the uterus.

Antiangiogenic effect of alpha-anordrin in vitro and in vivo.

AIM: To study the antiangiogenic effect of alpha-anordrin (alpha-Ano), a partial antagonist of estrogen receptor. METHODS: The in vivo inhibitory effect of alpha-Ano on angiogenesis was determined by microvascular density (MVD) in tumors and the chicken chorioallantoic membrane (CAM) model. The in vitro effects of alpha-Ano on proliferation, migration, and attachment of human umbilical vein endothelial cells (HUVEC) were assessed by trypan blue exclusion, wound-induced two-dimensional migration model, and their ability to adhere to type I collagen, respectively. The possible involvement of nitric oxide (NO) in alpha-Ano antiangiogenic effect was determined by measuring NO content using fluorescent assay. RESULTS: alpha-Ano significantly inhibited the MVD in Lewis lung carcinoma model and this effect was correlated with its inhibition of the tumor growth. alpha-Ano also showed an inhibitory effect on the angiogenesis of CAM with the inhibitory rate of 53% and such action of alpha-Ano could not be blocked by simultaneous administration of 17 beta-estrodiol, a typical agonist of estrogen receptor. In vitro studies showed that alpha-ANO obviously suppressed the proliferation and migration of HUVEC, but had no obvious effect on the attachment of HUVEC to the type I collagen. Moreover, alpha-Ano significantly reduced the level of NO released by HUVEC in a dose- and time-dependent manner. CONCLUSION: alpha-Ano possesses an antiangiogenic effect, and this effect is mediated, at least in part, by reducing the NO content and subsequently inhibiting the proliferation and migration of endothelial cells.

Effects of anordrin, droloxifene, nomegestrol, and mifepristone on cultured rat luteal cell apoptosis.

AIM: To study the effect of four kinds of antifertility agents anordrin(Ano), droloxifene(Dro), nomegestrol (Nom), and mifepristone (Mif) on luteal cell apoptosis. METHODS: Cultured rat luteal cells were incubated with different agents. HE stain was used to observe morphological changes. Extracted DNA was electrophoresed on agarose gel. Apoptotic cells were quantitated by flow cytometry. RESULTS: All 4 drugs reduced cell viability. Dro induced apoptosis while the other 3 drugs induced necrosis. Typical DNA ladders were observed after cells were incubated with Dro and there were 15.4%, 75.4%, or 90.5% apoptotic cells after treatment with Dro 1.25, 2.5, or 3.75 mg.L-1, respectively. CONCLUSION: Dro induced apoptosis while Ano, Nom, and Mif induced necrosis in cultured rat luteal cells.

[A randomized multicentre clinical trial on different doses of mifepristone alone and in combination with anordrin as emergency contraception].

OBJECTIVES: To investigate the effectiveness and side-effects of different doses of mifepristone alone or in combination with anordrin given orally within 96 hours after unprotected intercourse as an emergency contraceptive. METHODS: 2,400 cases of healthy women were recruited and allocated randomly in 4 groups: single dose of 25 mg mifepristone, 25 mg mifepristone plus 7.5 mg anordrin, 10 mg mifepristone plus 5 mg anordrin and 10 mg mifepristone alone. The efficacy rates of contraception were estimated according to Dixon method. RESULTS: The total expected number of pregnancy was 171.9 for 2,387 subjects enrolled in the study. The number of observed pregnancies related to method failure was 32 cases. The contraceptive effectiveness rates for the above 4 groups were 80.9%, 85.3%, 90.6% and 67.3%, respectively. For the group received 10 mg mifepristone plus 5 mg anordrin, pregnancy rate was 0.7% which was significantly lower than that in 10 mg mifepristone alone group (2.2%, P < 0.05). The incidences of withdrawal bleeding were 9.2%, 4.9%, 3.4% and 6.7% for the 4 group respectively. Two groups received mifepristone in combination with anordrin showed significant lower incidence of withdrawal bleeding than those in mifepristone alone groups. CONCLUSION: Single oral administration of 25 mg mifepristone alone, and the combination of 10 mg mifepristone plus 5 mg anordrin were safe and effective treatment regimen for emergency contraception with no serious adverse reaction and disturbance on menstrual pattern.