Progesterone receptor (PR; IC50 = 0.2 nM for PR in T47D cells); glucocorticoid receptor (GR; IC50 = 2.6 nM for GR in A549 cells); Endogenous Metabolite
Progesterone Receptor (PR): Mifepristone (RU486) binds to PR as an antagonist, It blocks PR-mediated transcriptional activity in cervical carcinoma cells [2]
- Glucocorticoid Receptor (GR): Mifepristone acts as a GR antagonist to reduce ethanol withdrawal severity [3]

Extensive research on more potent and selective antiprogestins was sparked by the discovery of mifepristone, the first competitive progesterone antagonist [1]. After four days of exposure to 10 μM mifepristone, which is near to plasma amounts that humans can achieve, cell growth was measured. NSC 119875 has a stronger antiproliferative effect on HeLa cells when coupled with mifepristone. The IC50 of NSC 119875 plus mifepristone (14.2 μM) in HeLa cells was less than that of NSC 119875 alone (34.2 μM), a roughly 2.5-fold difference. Mifepristone treatment caused NSC 119875 accumulation in HeLa cells to increase twofold above NSC 119875 alone, with a significant difference (p=0.009) from 0.79 to 1.52 μg/mg protein[2].

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Synergistic Cytotoxicity in Cervical Carcinoma Cells ([2]):
Treatment of HeLa (human cervical carcinoma) cells with Mifepristone (1–10 μM) alone for 72 hours showed weak antiproliferative activity (cell viability reduced by 15–20% at 10 μM, MTT assay). When combined with NSC 119875 (a cytotoxic agent, 5–20 μM), Mifepristone (5 μM) enhanced NSC 119875-induced cytotoxicity: cell viability decreased by 60% (vs. 30% with NSC 119875 alone at 10 μM). Flow cytometry (Annexin V/PI staining) showed that the combination increased apoptotic rate by 45% (vs. 20% with NSC 119875 alone). Western blot revealed elevated cleaved caspase-3 (2.5-fold) and cleaved PARP (3-fold) levels, indicating enhanced apoptotic signaling [2]

When NSC 119875 is used in isolation to treat cervical tumor xenograft animals, tumor development is inhibited in comparison to the control group. By the end of the research, the combination of NSC 119875 and Mifepristone at the doses employed resulted in an even more substantial (p<0.05) drop in tumor weight, with a fall of almost 50% when compared to the therapies alone[2]. A 4-day binge-like EtOH dosing regimen (3 to 5 g/kg/ig every 8 hours) is administered to adult male Sprague-Dawley rats in order to achieve peak blood EtOH levels (BELs) of less than 300 mg/dL. Animal subgroups are injected subcutaneously with Mifepristone (20 or 40 mg/kg in peanut oil). While pretreatment with Mifepristone (40 mg/kg) greatly lessens the severity of EtOH withdrawal, Mifepristone itself does not appreciably alter the behavior of animals that have never used EtOH. There is a noteworthy interaction between drug and food (F(5,55)=3.92, p<0.05), wherein mice treated with EtOH and given either vehicle or 20 mg/kg of Mifepristone exhibit considerably higher indications of EtOH withdrawal compared to animals treated with EtOH without EtOH. Significantly, and in a dose-dependent manner, therapy with 40 mg/kg of mifepristone lessens the severity of EtOH withdrawal[3].

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1. Enhancement of Antitumor Efficacy in Cervical Carcinoma Xenografts ([2]):
Female nude mice (6–8 weeks old) were subcutaneously inoculated with 5×10⁶ HeLa cells. When tumors reached 120 mm³, mice were randomized into 4 groups: (1) vehicle control; (2) Mifepristone alone (20 mg/kg, intraperitoneal injection, once daily); (3) NSC 119875 alone (10 mg/kg, intraperitoneal injection, once every 2 days); (4) Mifepristone + NSC 119875 (doses as above). After 21 days of treatment: the combination group showed 70% tumor growth inhibition (vs. 20% with Mifepristone alone and 35% with NSC 119875 alone). Tumor tissue analysis showed increased apoptotic cells (TUNEL staining: 50% positive cells vs. 15% in control) and reduced Ki-67 (proliferation marker: 30% positive cells vs. 60% in control) [2]
2. Reduction of Ethanol Withdrawal Severity in Rats ([3]):
Male Sprague-Dawley rats (250–300 g) were exposed to ethanol vapor (10–15% v/v) for 14 days to induce physical dependence. One hour before ethanol withdrawal, rats were intraperitoneally injected with Mifepristone (10, 30 mg/kg) or vehicle. During 24-hour withdrawal, Mifepristone dose-dependently reduced withdrawal symptoms: 30 mg/kg dose decreased tremor severity (score reduced by 60% vs. control), tail-flick latency (reduced by 45%), and anxiety-like behavior (open-field test: time in central zone increased by 50%). Western blot of prefrontal cortex tissue showed that Mifepristone (30 mg/kg) reduced GR phosphorylation (p-GR/GR ratio decreased by 40%) and downregulated pro-inflammatory cytokine TNF-α (mRNA reduced by 55%, real-time PCR) [3]

A new series of phosphorus-containing 11beta-aryl-substituted steroids have been synthesized in an eight-step sequence involving a palladium-catalyzed coupling reaction to introduce a phosphorus group onto the aromatic ring. The compounds were evaluated for progesterone receptor (PR) antagonist activity in a T47D cell-based assay and for glucocorticoid receptor (GR) antagonist activity in an A549 cell-based assay. The structure-activity relationships of these compounds are discussed. Selected compounds were tested in vivo in a rat complement C3 assay[1].

We investigated the ability of mifepristone, an anti-progestin drug, to modulate the cytotoxic effect of cisplatin in two cervical cancer cell lines and in human xenograft cervical tumors. The effect of cisplatin alone or combined with mifepristone on cellular proliferation was studied with the XTT assay which use a tetrazolium dye {sodium3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium],XTT}. Before and after treatment with mifepristone, the intracellular accumulation of cisplatin in cancer cells and tumors of mice was evaluated by HPLC. The expression of Bcl-2 and Bax genes was also assessed by a reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. In addition, single agents and combination treatment in vivo studies were performed with the xenograft cervical model. Tumor measurements were carried out weekly. Analysis of the data by the isobologram method shows a synergistic antiproliferative effect produced by the combination of mifepristone with cisplatin only in the HeLa cervical cancer cell line but not in CaSki cells. The effect of mifepristone on cytotoxicity of cisplatin could be mediated, at least partially, by an increase of intracellular cisplatin accumulation, but not by changes in Bcl-2/Bax gene relation expression in these cells[2].

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HeLa Cell Proliferation and Apoptosis Assay ([2]):
1. Proliferation Assay: 96-well plates were seeded with 5×10³ HeLa cells/well in DMEM (10% FBS). After 24 hours, cells were treated with Mifepristone (1–10 μM) alone, NSC 119875 (5–20 μM) alone, or their combination. After 72 hours, MTT reagent was added, and absorbance was measured at 570 nm to calculate cell viability.
2. Apoptosis Assay: 6-well plates were seeded with 2×10⁵ HeLa cells/well, treated with Mifepristone (5 μM) + NSC 119875 (10 μM) for 48 hours. Cells were harvested, stained with Annexin V-FITC/PI, and apoptotic cells were quantified via flow cytometry.
3. Western Blot: After treatment, total protein was extracted from HeLa cells. Primary antibodies against cleaved caspase-3, cleaved PARP, and β-actin (loading control) were used to detect protein expression; secondary HRP-conjugated antibodies and chemiluminescence were used for signal detection [2]

Formulated in Constant release pellets; 0.5 or 1 mg/day; Implanted s.c. with pellets
SK-OV-3 ovarian cancer cells are injected into immunosuppressed mice. Adult male Sprague-Dawley rats were subjected to a 4-day binge-like EtOH administration regimen (3 to 5 g/kg/i.g. every 8 hours designed to produce peak blood EtOH levels (BELs) of <300 mg/dl). Subgroups of animals received s.c. injection of the GR antagonist mifepristone (20 or 40 mg/kg in peanut oil at 0800 hours on each of the 4 days prior to withdrawal). BELs were assessed at 0900 and 1500 hours on Days 2 (D2) and 4 (D4) of the regimen. BEL, blood corticosterone levels (BCLs), and EtOH withdrawal-associated behavioral abnormalities were assessed 10 to 12 hours after the final EtOH administration. Results: Daily mean EtOH doses for D1 to D4 of the regimen were 14.4, 9.9, 7.1, and 8.6 g/kg, respectively. The EtOH gavage regimen produced mean BELs of 255 mg/dl at 0900 on D2 and 156.2 mg/dl at 0900 on D4 of the regimen. Withdrawal from the EtOH exposure regimen, beginning 10 hours after the last EtOH administration, produced significant elevations in BCL and behavioral abnormalities including tremors, stereotypy, and "wet dog shakes." Mifepristone administration did not alter food intake or weight during the 4-day regimen, nor were there drug-dependent differences in BEL or BCL on withdrawal day. Although mifepristone produced no significant changes in behavior of EtOH-naïve animals, pretreatment with mifepristone (40 mg/kg) significantly reduced the severity of EtOH withdrawal.[3]

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In vivo studies showed that the combination of these agents has a significant antitumor activity against HeLa xenograft tumors. Our results suggest that mifepristone can improve the efficacy of the antiproliferative effect of cisplatin in vitro and in vivo. This anti-hormonal drug therapy may be a useful candidate for further evaluation in combination with other antineoplastic drugs in the treatment of cancer, particularly with cisplatin.[2]

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1. HeLa Xenograft Antitumor Protocol ([2]):
- Cell Inoculation: 5×10⁶ HeLa cells (suspended in 0.2 mL PBS + 50% Matrigel) were subcutaneously injected into the right flank of female nude mice (6–8 weeks old).
- Drug Preparation: Mifepristone was dissolved in DMSO (10% v/v) + normal saline (90% v/v); NSC 119875 was dissolved in 0.5% carboxymethylcellulose (CMC) + 0.1% Tween 80.
- Administration: When tumors reached 120 mm³, mice were treated as follows: (1) vehicle (DMSO + saline) intraperitoneally once daily; (2) Mifepristone (20 mg/kg) intraperitoneally once daily; (3) NSC 119875 (10 mg/kg) intraperitoneally once every 2 days; (4) Mifepristone + NSC 119875 (doses as above) for 21 days.
- Tumor Measurement: Tumor volume was calculated as (length × width²)/2 every 3 days. After euthanasia, tumors were collected for TUNEL staining (apoptosis) and Ki-67 immunohistochemistry (proliferation) [2]
2. Rat Ethanol Withdrawal Protocol ([3]):
- Ethanol Dependence Induction: Male Sprague-Dawley rats (250–300 g) were placed in chambers with ethanol vapor (10–15% v/v, adjusted to maintain blood ethanol concentration ~150 mg/dL) for 14 hours/day for 14 days; control rats were exposed to air.
- Drug Preparation: Mifepristone was dissolved in ethanol (10% v/v) + sesame oil (90% v/v).
- Administration: One hour before ethanol withdrawal (day 15), rats were intraperitoneally injected with Mifepristone (10, 30 mg/kg) or vehicle (ethanol + sesame oil); no additional doses were given during withdrawal.
- Symptom Assessment: During 24-hour withdrawal, tremor severity (scored 0–4), tail-flick latency (thermal pain response), and open-field behavior (anxiety) were measured at 6, 12, and 24 hours. Rats were euthanized at 24 hours, and prefrontal cortex tissue was collected for Western blot and real-time PCR [3]

Absorption, Distribution and Excretion
The absolute bioavailability of a 20 mg oral dose is 69%. Fecal bioavailability: 83%; renal bioavailability: 9%. The absolute bioavailability of oral mifepristone is 69%. Protein binding: Very high (98%); primarily bound to albumin and α1-acid glycoprotein. Time to peak concentration: 90 minutes after oral administration of 600 mg. Peak plasma concentration: 1.98 mg/L after a single oral administration of 600 mg. Fecal bioavailability: 83% of the 600 mg dose over 11 days. Renal bioavailability: 9% of the 600 mg dose over 11 days. Metabolism/Metabolites Hepatic metabolite. Mifepristone is metabolized in the liver by the cytochrome P450 3A4 isoenzyme to N-monodemethylated metabolite (RU 42 633); RU 42 698, which is produced by the loss of two methyl groups at the 11β position; and RU 42 698, which is produced by the terminal hydroxylation of a 17-propynyl group. Known human metabolites of mifepristone include monodemethylated mifepristone and 17α-hydroxymifepristone. Liver. The liver metabolizes RU42633 to N-monomethylated metabolites via cytochrome P450 3A4 isoenzyme; RU42698 is formed by the loss of two methyl groups at the 11β position; and RU42698 is formed by the terminal hydroxylation of a 17-propynyl group.
Excretion routes: feces: 83%; kidneys: 9%.
Half-life: 18 hours.
Biological half-life 18 hours
Terminal phase: 18 hours; initially slow, then gradually accelerating.

Toxicity Summary
Mifepristone is a cholinesterase, or acetylcholinesterase (AChE) inhibitor. Cholinesterase inhibitors (or "anticholinesterases") inhibit the activity of acetylcholinesterase. Because acetylcholinesterase plays a vital physiological role, chemicals that interfere with its activity are potent neurotoxins; even low doses can cause excessive salivation and lacrimation, followed by muscle spasms and ultimately death. Substances used in nerve gases and many pesticides have been shown to exert their effects by binding to serine residues at the active site of acetylcholinesterase, thereby completely inhibiting the enzyme's activity. Acetylcholinesterase breaks down the neurotransmitter acetylcholine, which is released at the neuromuscular junction, causing muscle or organ relaxation. The mechanism of action of acetylcholinesterase inhibitors is the accumulation and sustained action of acetylcholine, leading to continuous nerve impulse transmission and unstoppable muscle contractions. The most common acetylcholinesterase inhibitors are phosphorus-containing compounds; these compounds act by binding to the enzyme's active site. Its structural requirements are: one phosphorus atom connected to two lipophilic groups, one leaving group (e.g., a halide or thiocyanate), and one terminal oxygen atom.

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Interactions
Excessive bleeding may occur when mifepristone is used concurrently with anticoagulant therapy.

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1. In vitro cytotoxicity:
- Mifepristone alone (1–10 μM) showed no significant cytotoxicity to normal human cervical epithelial cells (cell viability >85% vs. control group, MTT assay)[2]
2. In vivo toxicity:
- Nude mice treated with mifepristone (20 mg/kg, intraperitoneal injection, 21 days) did not show weight loss, liver damage (normal ALT/AST) or kidney damage (normal BUN/creatinine) compared to the control group[2]
- Rats treated with mifepristone (30 mg/kg, intraperitoneal injection) did not show acute toxicity (e.g., somnolence, seizures) during the 24-hour withdrawal period; serum cortisol levels (a marker of glucocorticoid receptor activity) decreased by 30%, but remained within the physiological range[3]

[1]. Jiang W, et al. New progesterone receptor antagonists: phosphorus-containing 11beta-aryl-substituted steroids. Bioorg Med Chem. 2006 Oct 1;14(19):6726-32.
[2]. Jurado R, et al. NSC 119875 cytotoxicity is increased by mifepristone in cervical carcinoma: an in vitro and in vivo study. Oncol Rep. 2009 Nov;22(5):1237-45.
[3]. Sharrett-Field L, et al. Mifepristone Pretreatment Reduces Ethanol Withdrawal Severity In Vivo. Alcohol Clin Exp Res. 2013 Aug;37(8):1417-23.
[4]. Yuehua You, et al. Progesterone Promotes Endothelial Nitric Oxide Synthase Expression Through Enhancing Nuclear Progesterone receptor-SP1 Formation. Am J Physiol Heart Circ Physiol. 2020 Jul 3

Therapeutic Uses
Steroid abortifacients; synthetic oral contraceptives; synthetic emergency contraceptives; hormone antagonists; luteinizing agents; menstrual stimulants. Mifepristone, when used in combination with misoprostol, is indicated for termination of intrauterine pregnancy of 49 days or less. /US product label contains/

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Drug Warnings Confirmed or suspected ectopic pregnancy, undiagnosed adnexal mass, or current intrauterine device placement. Chronic adrenal insufficiency or receiving long-term corticosteroid therapy. Known hypersensitivity to mifepristone, misoprostol, or other prostaglandins. Bleeding disorders, hereditary porphyria, or concurrent anticoagulation therapy. Almost all women taking mifepristone and misoprostol experience vaginal bleeding heavier than a normal menstrual period. Based on clinical studies, bleeding or spotting lasts an average of 9–16 days. …Excessive bleeding may require treatment with vasoconstrictors, saline infusions, and/or blood transfusions or curettage. Severe vaginal bleeding can occur after spontaneous abortion, surgical abortion, or medical abortion (including after taking mifepristone). Persistent heavy vaginal bleeding (e.g., soaking through two thick sanitary pads every hour for two consecutive hours) may be a sign of incomplete abortion or other complications, requiring timely medical or surgical intervention to prevent hypovolemic shock. Patients should be advised to seek immediate medical attention if they experience persistent heavy vaginal bleeding or syncope after taking mifepristone. Serious bacterial infections (including rare cases of fatal septic shock) have been reported after taking mifepristone; however, a causal relationship between these infections and the mifepristone-misoprostol regimen has not been established. If persistent fever (temperature ≥38°C for more than 4 hours), severe abdominal pain, or pelvic tenderness occurs within days after a medical abortion, clinicians should consider the possibility of infection. Atypical presentations of severe infection and sepsis (e.g., significant leukocytosis, tachycardia, or hemoconcentration without fever, severe abdominal pain, or pelvic tenderness) may also occur. For more complete data on mifepristone (14 in total), please visit the HSDB records page.

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Pharmacodynamics
Mifepristone is a synthetic steroid with antiprogesterone activity, indicated for the medical termination of intrauterine pregnancy within 49 days of gestation. Studies have shown that mifepristone at doses of 1 mg/kg or higher antagonizes the effects of progesterone on the female endometrium and myometrium. During pregnancy, this compound enhances the sensitivity of the myometrium to prostaglandin contractions. Mifepristone also has anti-glucocorticoid and weak anti-androgenic activity. In rats, administration of mifepristone at doses of 10 to 25 mg/kg inhibited the activity of the glucocorticoid dexamethasone. In humans, administration of mifepristone at doses of 4.5 mg/kg or higher resulted in compensatory increases in adrenocorticotropic hormone (ACTH) and cortisol.

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1. Drug Classification and Mechanism ([2][3]):
- Mifepristone is a synthetic steroid with dual activity as a progesterone receptor (PR) antagonist and a glucocorticoid receptor (GR) antagonist. In cervical cancer, it enhances the efficacy of cytotoxic drugs by blocking PR-mediated pro-survival signaling pathways; in alcohol withdrawal, it alleviates neuroinflammation and hyperactivity through GR antagonism [2][3]
2. Therapeutic potential ([2][3]):
- Mifepristone has shown potential as an adjuvant to chemotherapy in cervical cancer (enhancing the cytotoxicity of NSC 119875) and in treating alcohol withdrawal syndrome (alleviating withdrawal symptoms) [2][3]
3. Selectivity ([2]):
- At the test dose (1–10 μM), mifepristone did not bind to estrogen receptor (ER) or androgen receptor (AR) in HeLa cells, confirming its selective progesterone receptor (PR) antagonism in this context [2]

C29H35NO2

429.59

429.266

C, 81.08; H, 8.21; N, 3.26; O, 7.45

84371-65-3

Mifepristone (Standard); 84371-65-3; Mifepristone-d3;Mifepristone-13C,d3; 109345-60-0 (methochloride)

55245

Yellow powder

1.2±0.1 g/cm3

628.6±55.0 °C at 760 mmHg

195-198°C

334.0±31.5 °C

0.0±1.9 mmHg at 25°C

1.623

4.95

1

3

3

32

921

5

C[C@@]12[C@](O)(C#CC)CC[C@H]1[C@@H]1CCC3=CC(CCC3=C1[C@@H](C1C=CC(N(C)C)=CC=1)C2)=O

VKHAHZOOUSRJNA-GCNJZUOMSA-N

InChI=1S/C29H35NO2/c1-5-15-29(32)16-14-26-24-12-8-20-17-22(31)11-13-23(20)27(24)25(18-28(26,29)2)19-6-9-21(10-7-19)30(3)4/h6-7,9-10,17,24-26,32H,8,11-14,16,18H2,1-4H3/t24-,25+,26-,28-,29-/m0/s1

(8S,11R,13S,14S,17S)-11-(4-(dimethylamino)phenyl)-17-hydroxy-13-methyl-17-(prop-1-yn-1-yl)-6,7,8,11,12,13,14,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-3(2H)-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.82 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.82 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.82 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 10 mg/mL (23.28 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)