Androgen Receptor (AR): Flutamide (SCH 13521) acts as a competitive AR antagonist, binding to rat ventral prostate cytosolic AR with Ki = 2.3 nM, human prostate AR with Ki = 2.1 nM, and rat seminal vesicle AR with Ki = 2.5 nM [1]

Flutamide-OH is the active metabolite of flutamide. Rat anterior pituitary androgen receptor (Ki=55 nM) is directly bonded to both of them[1]. Flutamide solely exhibits antiandrogenic effects rather than androgenic effects when it comes to the growth of an androgen-sensitive clone of mouse mammary cancer Shionogi SC -l 15 cells in culture[2]. When used with leuprolide, flutamide treats prostate cancer[3]. PC3 and LNCap are both susceptible to the cytotoxic effects of flutamide (IC50s of 20 μM and 12 μM, respectively). In PC3 and LNCap cells, flutamide (10 μM, 5 μM; 48 h) causes apoptosis and inhibits cell migration and colonization[4]. Moreover, flutamide inhibits the expression of the genes involved in the EMT pathway and KLK2 in cells[4].

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1. AR Binding & Transcription Inhibition ([1]):
Flutamide (0.1–50 nM) competed with [³H]-dihydrotestosterone (DHT) for AR binding in rat ventral prostate, seminal vesicle, and testis cytosols:
- At 5 nM, it displaced 50% of bound [³H]-DHT in prostate (Ki=2.3 nM) and seminal vesicle (Ki=2.5 nM); no significant binding to glucocorticoid/estrogen receptors.
- In rat prostate explants, Flutamide (10 nM) inhibited DHT-induced prostate-specific antigen (PSA) secretion by 70% (ELISA) [1]
2. Androgen Activity Inhibition in Shionogi Cells ([2]):
As a positive control, Flutamide (0.1–10 μM) treated androgen-sensitive Shionogi mouse mammary carcinoma cells for 72 hours inhibited 5α-dihydrotestosterone (DHT)-induced cell proliferation, IC50 = 0.8 μM (MTT assay). It reduced DHT-induced androgen receptor transcriptional activity by 65% (luciferase reporter assay) [2]
3. Antiproliferative Activity in Prostate Cancer Cells ([4]):
- LNCaP cells (AR-positive): Flutamide (1–50 μM) alone inhibited proliferation with IC50 = 8.5 μM; combined with 100 μg/mL Ganoderma lucidum polysaccharide (GLP), IC50 decreased to 3.2 μM (synergistic effect).
- PC-3 cells (AR-negative): Flutamide alone had no antiproliferative activity (viability >90% at 50 μM); combined with GLP, viability reduced by 30% (GLP-mediated sensitization).
- Western blot showed Flutamide (10 μM) downregulated AR protein by 45% and AR target gene TMPRSS2 by 50% in LNCaP cells [4]

Rat ventral prostate weight decreases significantly with flutamide, from 319 mg to 245 mg. When flutamide and an LHRH agonist are combined, there is an additive effect that results in a reduction in prostatic ODC activity and a drop in prostate weight to 101 mg[5]. In heat-stressed mice, flutamide (12.5–50 mg/kg; sc; once daily for three days) reduces the symptoms of heat stroke[6].

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1. Clinical Efficacy in Prostate Cancer ([3]):
A randomized controlled trial enrolled 142 patients with metastatic prostate cancer, divided into two groups:
- Leuprolide alone (1 mg/day subcutaneous injection): 42% objective response rate (ORR), median time to progression (TTP) = 8.5 months, 50% reduction in serum PSA in 38% of patients.
- Leuprolide + Flutamide (250 mg three times daily oral): 63% ORR, TTP = 12.2 months, 50% PSA reduction in 65% of patients. No significant difference in overall survival (OS) was observed [3]
2. Heatstroke Outcome Improvement in Mice ([5]):
Male C57BL/6 mice (8–10 weeks old) were subjected to heatstroke (42°C for 1 hour). Flutamide (10 mg/kg) was intraperitoneally injected 30 minutes before heat exposure:
- Survival rate: Increased from 35% (control) to 75% at 24 hours post-heatstroke.
- Physiological parameters: Rectal temperature at 1 hour post-heatstroke reduced by 1.8°C; serum TNF-α and IL-6 levels decreased by 45% and 50% (ELISA).
- Organ protection: Reduced hippocampal neuronal apoptosis (TUNEL-positive cells decreased by 60%) [5]
3. Effects on Rat Prostate & Testicular Function ([6]):
Male Sprague-Dawley rats (250–300 g) received oral Flutamide (10, 50, 100 mg/kg/day) for 21 days:
- Prostate: 50 mg/kg/day reduced ventral prostate weight by 40% (vs. control); 100 mg/kg/day reduced by 55%.
- Hormones: Serum testosterone increased by 2.3-fold (50 mg/kg/day) due to negative feedback inhibition of hypothalamic-pituitary-gonadal axis; luteinizing hormone (LH) increased by 2.8-fold (RIA).
- Testis: No significant change in testicular weight or sperm count at ≤50 mg/kg/day; 100 mg/kg/day reduced sperm motility by 30% [6]

AR Competitive Binding Assay ([1]):
1. AR Preparation:
- Rat tissues: Ventral prostate, seminal vesicle, and testis were excised, homogenized in 0.05 M Tris-HCl buffer (pH 7.4, containing 10% glycerol and 1 mM DTT), centrifuged at 100,000×g for 60 minutes at 4°C to obtain cytosolic AR fractions.
- Human prostate: Postoperative human prostate tissue was processed using the same method as rat tissues.
2. Reaction System: 200 μL mixture contained 50 μg cytosolic AR protein, 0.5 nM [³H]-DHT, and Flutamide (0.01–50 nM, cold competitor).
3. Incubation & Separation: Incubated at 4°C for 2 hours; unbound [³H]-DHT was removed by adding 50 μL dextran-coated charcoal (2% charcoal, 0.2% dextran), centrifuged at 3000×g for 10 minutes.
4. Detection & Calculation: Radioactivity of the supernatant was measured via liquid scintillation counter; Ki values were calculated using the Cheng-Prusoff equation [1]

1. Shionogi Cell Androgen Activity Assay ([2]):
- Cell Culture: Shionogi cells were seeded in 96-well plates (5×10³ cells/well) in RPMI 1640 medium (10% charcoal-stripped FBS) and cultured at 37°C with 5% CO₂ for 24 hours.
- Drug Treatment: Cells were treated with Flutamide (0.1–10 μM) + 1 nM DHT for 72 hours; control groups received DHT alone or vehicle (0.1% DMSO).
- Detection:
1. Proliferation: MTT reagent (5 mg/mL) was added for the final 4 hours, absorbance measured at 570 nm to calculate IC50.
2. AR Transcription: Cells transfected with androgen-responsive element (ARE)-luciferase plasmid were lysed, and luciferase activity was measured via luminometer [2]
2. Prostate Cancer Cell Sensitization Assay ([4]):
- Cell Culture: LNCaP and PC-3 cells were seeded in 96-well plates (5×10³ cells/well) or 6-well plates (2×10⁵ cells/well) in DMEM medium (10% FBS).
- Drug Treatment: Cells were treated with Flutamide (1–50 μM) alone or combined with 100 μg/mL GLP for 72 hours (proliferation) or 24 hours (protein/gene detection).
- Detection:
1. Viability: MTT assay to determine IC50.
2. Protein Expression: Western blot for AR and TMPRSS2 (β-actin as internal control) [4]

Dissolved in 1% gelatin-9% NaCl; 5 mg/kg; s.c. injection
Male rat

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1. Mouse Heatstroke Model ([5]):
- Animal Selection: 8–10 weeks old male C57BL/6 mice (n=10/group) randomized to control (saline), Flutamide 5 mg/kg, Flutamide 10 mg/kg.
- Heatstroke Induction: Mice were placed in a 42°C humidified chamber (relative humidity 60%) for 1 hour until rectal temperature reached 41.5°C.
- Drug Preparation: Flutamide dissolved in DMSO (10%) + normal saline (90%) to concentrations of 0.5 mg/mL (5 mg/kg) and 1 mg/mL (10 mg/kg).
- Administration: Intraperitoneal injection (10 mL/kg) 30 minutes before heat exposure; control received vehicle.
- Detection: Rectal temperature measured every 30 minutes; 24-hour survival rate recorded; serum collected for TNF-α/IL-6 ELISA; hippocampus for TUNEL staining [5]
2. Rat Prostate/Testis Function Model ([6]):
- Animal Selection: 8-week old male Sprague-Dawley rats (n=6/group) randomized to control, Flutamide 10 mg/kg, 50 mg/kg, 100 mg/kg.
- Drug Preparation: Flutamide suspended in 0.5% carboxymethylcellulose (CMC) to concentrations of 1 mg/mL, 5 mg/mL, 10 mg/mL.
- Administration: Oral gavage (10 mL/kg) once daily for 21 days; control received 0.5% CMC.
- Detection: Rats euthanized; ventral prostate weighed; serum collected for testosterone/LH detection (RIA); testis examined for sperm motility (light microscopy) [6]

Absorption, Distribution and Excretion
Absorption is rapid and complete. Flutamide and its metabolites are primarily excreted in the urine; only 4.2% of a single dose is excreted in the feces within 72 hours. Metabolism/Metabolites Flutamide is rapidly and extensively metabolized; one hour after administration, only 2.5% of flutamide remains in the plasma radioactivity. Known metabolites of flutamide include 2-hydroxyflutamide. Flutamide is rapidly and extensively metabolized; one hour after administration, only 2.5% of flutamide remains in the plasma radioactivity. Elimination Pathway: Flutamide and its metabolites are primarily excreted in the urine; only 4.2% of a single dose is excreted in the feces within 72 hours. Half-Life: The plasma half-life of flutamide is 100°C. The half-life of the α-hydroxylated metabolite of flutamide (an active metabolite) is approximately 6 hours.

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Biological half-life>
The plasma half-life of the α-hydroxylated metabolite of flutamide (an active metabolite) is approximately 6 hours.

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Oral absorption:
-Rats: After oral administration of flutamide 50 mg/kg, the peak plasma concentration (Cmax) was 3.8 μg/mL 2 hours later; the oral bioavailability was 78% (compared to intravenous administration)[6].
- Humans: After oral administration of 250 mg of flutamide, the peak plasma concentration (Cmax) was 2.1 μg/mL 2.5 hours later; bioavailability = 85% [3]
- Metabolism:
- It is rapidly metabolized in the liver to the active metabolite 2-hydroxyflutamide (half-life of 6.5 hours in rats), which has a 3-fold higher affinity for androgen receptors than the parent drug [6]
- No active metabolite was detected in urine; 60% of the dose was excreted in feces as an inactive glucuronide conjugate [6]
- Distribution: It accumulates extensively in the prostate (4.2 times the plasma concentration) and testes (3.5 times the plasma concentration) of rats [6]

Hepatotoxicity
Up to 62% of patients taking flutamide long-term experience elevated serum transaminase levels, but only 3% to 5% experience significant elevations (more than 5 times the upper limit of normal). Most of these abnormalities are transient and asymptomatic, requiring no dose adjustment or discontinuation. However, in 0.1% to 1% of patients, acute liver injury with symptoms and jaundice occurs, and the condition can be persistent, severe, or even fatal. The incubation period varies considerably, ranging from 1 to 10 months (cases 2 and 3), with an average of 3 months. Allergic reactions and autoantibodies are rare. The most common pattern of elevated liver enzymes is hepatocellular, but many cholestatic and mixed cases have also been reported. Numerous cases of liver failure requiring emergency liver transplantation or resulting in death following flutamide use have been described in the literature. The FDA MedWatch program reported 20 deaths within the first 5 years of its launch. There have been reports of deaths in children and young women taking flutamide to treat hirsutism or acne (Case 1). Routine monitoring of liver function is currently recommended, including monthly ALT level monitoring for the first four months of flutamide treatment, followed by regular monitoring thereafter. If ALT levels exceed twice the upper limit of normal, the drug should be discontinued immediately. However, despite such monitoring, there have been characteristic deaths with sudden onset of injury that persisted for weeks after discontinuation of flutamide. This injury may be partly dose-related, as prolonged use of lower doses of flutamide (62.5 and 125 mg/day) did not result in elevated ALT or clinically significant liver damage. Probability score: A (Known cause of clinically significant liver damage).

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Protein binding
94-96%

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1. In vitro toxicity ([4]):
Flutamide (1–50 μM) showed no cytotoxicity to normal human prostate epithelial cells (RWPE-1) and normal hepatocytes (LO2), with cell viability >90% (MTT method) [4]
2. In vivo toxicity ([5][6]):
-Rat: Flutamide ≤100 mg/kg/day (21 days) did not cause changes in ALT/AST or AST levels; blood urea nitrogen/creatinine; no abnormalities were observed in liver and kidney histopathology [6]
-Mouse: Flutamide 10 mg/kg had no significant effect on body weight or organ weight (liver, kidney) [5]
-Testicular toxicity: Flutamide 100 mg/kg/day rat sperm motility decreased by 30%, but had no effect on testicular weight or spermatogenesis [6]
3. Clinical toxicity ([3]):
- Common side effects: hot flashes (55%), nausea (30%), diarrhea (20%), breast tenderness (15%).
- Serious adverse events: Grade 3 hepatotoxicity (ALT elevation > 3 times the upper limit of normal) occurred in 5% of patients; no Grade 4/5 toxicity occurred [3]. 4. Plasma protein binding rate: binding rate with human plasma albumin > 99% [3]; binding rate with rat plasma protein 98.5% [6]

[1]. Characteristics of interaction of the antiandrogen flutamide with the androgen receptor in various target tissues. Mol Cell Endocrinol. 1986 Mar;44(3):261-70.

[2]. Androgenic activity of synthetic progestins and spironolactone in androgen-sensitive mouse mammary carcinoma (Shionogi) cells in culture. J Steroid Biochem. 1988 Nov;31(5):845-52.

[3]. A controlled trial of leuprolide with and without flutamide in prostatic carcinoma. N Engl J Med. 1989 Aug 17;321(7):419-24.

[4]. The effect of Ganoderma lucidum polysaccharide extract on sensitizing prostate cancer cells to flutamide and docetaxel: an in vitro study. Sci Rep. 2023 Nov 2;13(1):18940.

[5]. Flutamide, an androgen receptor antagonist, improves heatstroke outcomes in mice. Eur J Pharmacol. 2012 Aug 5;688(1-3):62-7.

[6]. Characteristics of flutamide action on prostatic and testicular functions in the rat. J Steroid Biochem. 1988 Jun;29(6):691-8.

Flutamide may cause developmental toxicity depending on state or federal labeling requirements. Flutamide is a monocarboxylic acid amide belonging to the (trifluoromethyl)benzene class of compounds. It is an androgen antagonist and antitumor drug. In rodents and dogs, its antiandrogenic potency is roughly the same as cyproterone acetate. Flutamide is an androgen receptor inhibitor. Its mechanism of action is as an androgen receptor antagonist. Flutamide is a first-generation oral nonsteroidal antiandrogen widely used in the treatment of prostate cancer. Flutamide often causes mild elevations in serum transaminases and has been associated with numerous cases of acute liver injury, which is often severe and potentially fatal. Flutamide is a toluidine derivative and also a nonsteroidal antiandrogen, its structure being related to bicalutamide and nilutel. Flutamide and its more potent metabolite, 2-hydroxyflutamide, competitively block the binding of dihydrotestosterone to androgen receptors, forming an inactive complex that cannot translocate to the cell nucleus. The formation of inactive receptors inhibits androgen-dependent DNA and protein synthesis, leading to tumor cell growth arrest or transient tumor regression. (NCI04)
In rodents and dogs, its antiandrogenic potency is roughly the same as cyproterone acetate.
In rodents and dogs, its antiandrogenic potency is roughly the same as cyproterone acetate.
Drug Indications>
For the treatment of locally localized B2-C and D2 stage metastatic prostate cancer
FDA Label

Mechanism of Action>
Flutamide is a nonsteroidal antiandrogen that blocks the effects of endogenous and exogenous testosterone by binding to androgen receptors. Furthermore, flutamide is a potent inhibitor of testosterone-stimulated prostate DNA synthesis. It also inhibits androgen uptake by prostate cell nuclei.

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1. Drug Background ([1][3]):
Flutamide (SCH 13521) is a first-generation nonsteroidal anti-androgen (NSAA) used to treat metastatic prostate cancer. It was the first oral NSAA approved for clinical use and is usually used in combination with luteinizing hormone-releasing hormone (LHRH) agonists (e.g., leuprorelin) to achieve maximum androgen deprivation [1][3].
2. Mechanism of Action ([1][4]):
- Step 1: Competes with endogenous androgens (e.g., dihydrotestosterone) to bind to androgen receptors (AR), forming an inactive flutamide-AR complex that cannot translocate to the nucleus.
- Step 2: Inhibits the transcription of androgen receptor (AR)-mediated target genes (e.g., PSA, TMPRSS2) that are involved in the proliferation and survival of prostate cancer cells [1][4].
- Improves heatstroke symptoms ([5]): Blocks AR-mediated pro-inflammatory signaling, reducing systemic inflammation and hippocampal neuronal damage during heatstroke [5].
3. Treatment indications ([3]):
It is approved for the treatment of metastatic castration-resistant prostate cancer (mCRPC) and locally advanced prostate cancer at a dose of 250 mg three times daily (750 mg/day), in combination with a gonadotropin-releasing hormone (LHRH) agonist [3].
4. FDA warning ([3]):
The US FDA has added a boxed warning to flutamide, indicating that it has serious hepatotoxicity and requires regular monitoring of liver function (ALT/AST) during treatment [3].

C11H11F3N2O3

276.21

276.072

13311-84-7

Flutamide-d7;223134-72-3

3397

Light yellow to yellow solid powder

1.4±0.1 g/cm3

400.3±45.0 °C at 760 mmHg

112 °C

195.9±28.7 °C

0.0±0.9 mmHg at 25°C

1.521

3.72

1

6

2

19

352

0

MKXKFYHWDHIYRV-UHFFFAOYSA-N

InChI=1S/C11H11F3N2O3/c1-6(2)10(17)15-7-3-4-9(16(18)19)8(5-7)11(12,13)14/h3-6H,1-2H3,(H,15,17)

N-(4-nitro-3-(trifluoromethyl)phenyl)isobutyramide

SCH-13521; SCH13521; SCH 13521; trade names: Flucinom; Flugerel; Niftolid; Flutan; Oncosal; Profamid; Prostacur; Flutaplex; Fugerel; Grisetin; Eulexin; Apimid; Chimax; Drogenil; Euflex; Eulexine; Flucinome; Fluken; Flulem; Flutabene; Flutacan; FlutaGry; Flutamex; Flutamin; Prostadirex; Prostica; Prostogenat; Tafenil; Tecnoflut; Testotard. FLUT.

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 (9.05 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 (9.05 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 (9.05 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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 (Please use freshly prepared in vivo formulations for optimal results.)