AR/androgen-receptor (IC50 = 36 nM in LNCaP cells)[1]
Androgen Receptor (AR): Enzalutamide (MDV3100) binds to human AR with high affinity, competing with dihydrotestosterone (DHT) for binding; Ki = 0.15 nM. It also inhibits AR nuclear translocation and DNA binding to AR-responsive elements (AREs) [1]

Enzalutamide (MDV3100) in castration-resistant LNCaP/AR cells had a greater affinity for AR than ICI 176334 (AR-overexpressing) in a competitive assay using 16β-[18F]fluoro-5α-DHT (18-FDHT). On LNCaP/AR prostate cells, ezetimibe had no agonistic effects. Enzalutamide inhibits the activation of transmembrane serine protease 2 (TMPRSS2) and prostate-specific antigen (PSA) by binding to the synthetic androgen R1881, which is present in parental LNCaP cells. The transcriptional activity of the mutant AR protein (W741C, Trp741 altered to Cys) is inhibited by enzalutamide [1]. Additionally, enzalutamide inhibits the recruitment of coactivators and the nuclear translocation of ligand-receptor complexes [2].

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1. Antiproliferative Activity in CRPC Cells ([1][3]):
- Treatment of LNCaP (androgen-dependent) and C4-2 (castration-resistant prostate cancer, CRPC) cells with Enzalutamide (0.1–20 μM) for 72 hours inhibited proliferation: IC50 = 1.8 μM (LNCaP) and 2.5 μM (C4-2) (MTT assay) [1]. At 10 μM, it downregulated AR target genes: PSA mRNA (70% reduction, real-time PCR) and TMPRSS2 protein (65% reduction, Western blot) in C4-2 cells [1]
- In C4-2 cell clone formation assay, Enzalutamide (5 μM) reduced colony number by 80% vs. control (14-day culture, crystal violet staining). It also blocked AR nuclear accumulation: nuclear AR protein decreased by 75% (immunofluorescence) [3]

When administered at a dose of 10 mg/kg to castrated male mice harboring LNCaP/AR xenografts, enzalutamide (MDV3100) significantly reduces tumor growth [1]. The pharmacokinetics of enzalutamide show dose-independency when administered orally at dosages ranging from 0.5 to 5 mg/kg [4].

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MDV3100/enzalutamide and RD162 are orally available and induce tumor regression in mouse models of castration-resistant human prostate cancer. Of the first 30 patients treated with MDV3100 in a Phase I/II clinical trial, 13 of 30 (43%) showed sustained declines (by >50%) in serum concentrations of prostate-specific antigen, a biomarker of prostate cancer. These compounds thus appear to be promising candidates for treatment of advanced prostate cancer.[1]

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Enzalutamide Treatment Decreased Tumor Volume, Increased Body Weight, and Induced Apoptosis in a Mouse LNCaP-AR Xenograft Model[2]
To study the effects of enzalutamide and bicalutamide in vivo, a mouse xenograft CRPC model was developed using castrated male animals implanted with human LNCaP-AR cells that over-express wild-type AR 25. Animals were administered enzalutamide (1–50 mg/kg/day) or bicalutamide (50 mg/kg/day), and tumor volume and mouse body weight were measured at 2- to 3-day intervals for 28 days. Bicalutamide (50 mg/kg/day) inhibited tumor growth through Day 16 when compared with the vehicle control group. After Day 16, however, tumors in these mice grew continuously up to 154% of baseline by Day 28 (Fig. 3A, Table I). In contrast, enzalutamide (10 mg/kg/day) inhibited tumor growth significantly during the first 6 days of treatment compared with vehicle- and bicalutamide-treated mice (mean ± SE percentage tumor growth relative to baseline: vehicle, 119 ± 5%; enzalutamide 10 mg/kg, 86 ± 6%, bicalutamide 50 mg/kg, 106 ± 8%). By Day 13, enzalutamide treatment resulted in a 19% decrease in tumor volume at doses of 10 mg/kg/day or greater compared with the initial tumor size (Fig. 3A). Some tumors in the enzalutamide-treated groups (1 and 50 mg/kg) decreased in size significantly so as to be beyond the measurement limits (Table I, non-measurable tumors/group). These tumors were not included in further analysis. Tumor volume continued to decrease through Day 24 for the 10 mg/kg/day-enzalutamide group and through the last measured time point at Day 28 for the 50-mg/kg/day group (Table I). Maximal effect on tumor regression relative to initial tumor volumes in each group occurred at Day 28 or beyond of enzalutamide treatment (Fig. 3A). The mean ± SE relative tumor volume decline after 27 days of enzalutamide treatment was 41 ± 7% at 10 mg/kg and 68 ± 13% at 50 mg/kg compared with baseline. In contrast, after 27 days of vehicle or 50 mg/kg bicalutamide treatments, tumor volume increased 54% when compared with the baseline (Table I).

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Pharmacokinetic analyses[4]
The areas under the plasma concentration–time curve (AUC) and the first moment curve (AUMC) were calculated using the linear trapezoidal method, extrapolated to time = infinity. The terminal half-life (T½) was calculated as 0.693/λ, where λ is the slope of the log-linear portion of the concentration–time profile. The systemic clearance (CL), mean residence time (MRT), and the volume of the distribution at steady state (Vss) were calculated as dose/AUC, AUMC/AUC, and MRT·CL, respectively. The extent of absolute oral bioavailability (F) was estimated by dividing the AUC after oral administration by the AUC after intravenous administration of the respective dose. The peak concentration (Cmax) and the time to reach Cmax (Tmax) were read directly from individual plasma concentration–time profiles. The tissue-to-plasma partition coefficient (Kp) for enzalutamide was calculated by dividing the mean AUCtissue by the mean AUCplasma after administration. To obtain the pharmacokinetic parameters above, all plasma and tissue concentration–time profiles were analyzed using a non-compartmental method with non-linear least squares regression using the WinNonlin software

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1. Antitumor Efficacy in Mouse CRPC Models ([3]):
Male SCID mice (6–8 weeks old) were subcutaneously inoculated with 5×10⁶ C4-2 cells. When tumors reached 100 mm³, mice received oral Enzalutamide (10, 30 mg/kg/day) or vehicle for 28 days. The 30 mg/kg dose reduced tumor volume by 70% and tumor weight by 65% vs. control (tumor volume = length×width²/2, measured twice weekly). Tumor tissue analysis: Ki-67 (proliferation marker) positive rate decreased by 60% (immunohistochemistry), and AR target gene PSA mRNA reduced by 75% (real-time PCR) [3]
2. Clinical Activity in CRPC Patients ([2]):
In a phase 1–2 study of 140 CRPC patients (post-chemotherapy), oral Enzalutamide (160 mg/day) was administered until disease progression. After 12 weeks: (1) 78% of patients achieved ≥50% reduction in serum PSA (prostate-specific antigen, AR target); (2) 22% had objective tumor regression (RECIST criteria); (3) Median progression-free survival was 15.8 months. No significant antitumor activity was observed in patients with AR-null tumors [2]

Estimation of hepatic intrinsic clearance of enzalutamide in rat liver microsomes[4]
Rat liver microsomes were used to estimate the intrinsic clearance of enzalutamide. A typical reaction mixture (500 µL) consisted of rat liver microsomal protein (final concentration = 0.5 mg protein/mL incubation mixture) and an NADPH regenerating system (final concentrations: 1.3 mM NADP+, 3.3 mM glucose-6-phosphate, 0.4 U/mL glucose-6-phosphate dehydrogenase, and 3.3 mM magnesium chloride) in 100 mM potassium phosphate buffer (pH 7.4). The mixture was pre-incubated in a water bath at 37 °C for 5 min and an aliquot of enzalutamide solution added to a final concentration of 2 µM. Aliquots (50 µL) of the mixture were sampled at 0, 5, 15, and 30 min after initiation of the reaction. Immediately after collection, a stop solution (100 µL ice-chilled methanol) was added to the sample to terminate the reaction. After vigorous vortexing, and centrifuging at 10,000×g for 5 min, an aliquot (50 µL) of the supernatant was assayed. The concentration of enzalutamide remaining in the sample was plotted against the reaction time to determine the metabolic rate constant of the reaction.

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Estimation of the fraction of enzalutamide bound to plasma protein[4]
We conducted a protein-binding study to determine the fraction of unbound enzalutamide in rat plasma. Binding of test material was assessed via equilibrium dialysis using RED® devices. All assessments were made in triplicate. After 200 µL samples of plasma containing 2 µg/mL enzalutamide were placed into a sample chamber, 350 µL of phosphate buffer (pH 7.4) was added to the buffer chamber. The device containing the samples was incubated at 37 °C for 4 h in a shaking water bath. After incubation, enzalutamide in was assayed in plasma and buffer.

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AR Competitive Binding Assay ([1]):
1. Recombinant AR Preparation: Human AR ligand-binding domain (LBD) was expressed in E. coli and purified via nickel-affinity chromatography.
2. Reaction System: 200 μL mixture contained 50 mM Tris-HCl (pH 7.4), 10% glycerol, 0.5 nM [³H]-DHT (AR ligand), 100 ng AR-LBD, and Enzalutamide (0.01–10 nM, cold competitor).
3. Incubation & Separation: Incubated at 4°C for 2 hours; unbound [³H]-DHT was removed by adding dextran-coated charcoal (1% charcoal, 0.1% dextran) and centrifuging at 3000×g for 10 minutes.
4. Detection & Calculation: Radioactivity of supernatant was measured via liquid scintillation counter; Ki value was calculated using the Cheng-Prusoff equation [1]

Nuclear Translocation Assay[3]
The yellow fluorescent protein (YFP)-AR plasmid was donated by Marc I. Diamond and was stably transfected into HEK293 cells. Cells were seeded at 1.5 × 105 cells/cm2 in optical microplates in phenol red-free DMEM/F12 medium supplemented with 10% hormone-depleted FBS. After 2 days of cultivation, the cells were pre-treated with enzalutamide (1 µM) or bicalutamide (1 µM) for 2 hr and then co-treated with 1 nM DHT for 1 hr in the presence of enzalutamide or bicalutamide. Cells were then washed in phosphate-buffered saline, incubated with the nuclear fluorescent marker DAPI (1 µg/ml) for 30 min and fixed with 4% paraformaldehyde for 30 min at room temperature. Cells were visualized using a Qimaging digital camera coupled to an Olympus X71 fluorescence microscope using a YFP filter. Nuclear and total cellular AR-YFP fluorescence intensities (integrated density) were quantified using ImageJ software. Nuclear AR-YFP fluorescence was quantified in areas defined by image segmentation based on DAPI fluorescence, and the nuclear:total intensity ratio calculated. At least 14 cells were quantified per condition per independent experiment (n = 3). For live cell imaging experiments, cells were pre-treated with enzalutamide (1 or 10 µM) or bicalutamide (1 or 10 µM) for 2 hr and then co-treated with 1 nM DHT for 3 hr in the presence of enzalutamide or bicalutamide. Cells were imaged immediately before DHT addition (t′ = 0) and then at 60-min intervals over 3 hr.

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1. CRPC Cell Proliferation & Gene Assay ([1]):
- Cell Culture: LNCaP (androgen-dependent) and C4-2 (CRPC) cells were cultured in RPMI 1640 (10% FBS) and seeded in 96-well plates (5×10³ cells/well, proliferation) or 6-well plates (2×10⁵ cells/well, gene/protein).
- Drug Treatment: Cells were treated with Enzalutamide (0.1–20 μM) for 72 hours (proliferation) or 24 hours (gene/protein); control received 0.1% DMSO.
- Detection:
1. Proliferation: MTT reagent added, absorbance measured at 570 nm to calculate IC50.
2. Gene/Protein: Real-time PCR (PSA/TMPRSS2 mRNA) and Western blot (PSA, AR protein; β-actin as internal control) [1]
2. C4-2 Cell Clone Formation Assay ([3]):
- Cell Culture: C4-2 cells were seeded in 6-well plates (1×10³ cells/well) and cultured in RPMI 1640 (10% FBS) for 24 hours.
- Drug Treatment: Cells were treated with Enzalutamide (1, 5, 10 μM) for 14 days; medium + drug was refreshed every 3 days.
- Detection: Colonies were fixed with 4% paraformaldehyde, stained with crystal violet, and counted under a microscope. Colony formation rate = (colony number in drug group / control group) × 100% [3]

Formulated in 1% carboxymethyl cellulose, 0.1% Tween-80, 5% DMSO; 10 mg/kg; Oral gavage
Castration-resistant LNCaP/HR xenografts in male SCID mice Mouse Xenograft Model[3]
Following a 5-day acclimation period, 5- to 9-week-old male CB17SCID mice were castrated and allowed to recover for an additional 5 days before inoculation with tumor cells. LNCaP cells co-expressing exogenous AR and the AR-dependent reporter construct ARR2-Pb-Luc were used to generate a xenograft model of human prostate cancer. Before implantation, LNCaP-AR-Lux cells were prepared by the addition of trypsin-EDTA, washed with complete medium, collected and resuspended at 20 × 106 cells/ml. Cell suspensions were diluted with Matrigel to 2 × 106 cells/0.2 ml and delivered subcutaneously in the suprascapular region. Tumor growth was monitored to the volume of 100 mm3 when treatment began (∼80 days). The observed rate of tumor take with LNCaP-AR-Lux cells is between 70% and 80%. Body weight and tumor volumes (width2 × length/2) were measured two to three times per week with a digital caliper, and the average tumor volumes were determined. Test drugs were diluted in Tween 80:PEG 400, and stored at 4°C until administration by oral gavage. Each group of mice (n = 7) was treated daily for 28 consecutive days with 1, 10, or 50 mg/kg enzalutamide, vehicle control, or 50 mg/kg bicalutamide. At the end of the treatment period or when tumor volume exceeded 1,000 mm3, animals were euthanized and blood and tissue samples were collected for analysis.

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Intravenous and oral administration of enzalutamide in rats[4]
Enzalutamidewas dissolved in vehicle (10 % DMSO, 45 % polyethylene glycol 400, and 45 % saline) . As a single dose, the administration routes were an intravenous bolus via the tail vein (n = 3) and an oral gavage dose (n = 4). Dosing volume was 1 mL per kg (body weight) and the dosing range was 0.5, 2, and 5 mg/kg. The use of 2 mg/kg was reported in a previous study (Song et al. 2014). A blood sample (200 µL) was collected from the jugular vein using a heparinized syringe to ensure anticoagulation at 0.08 (intravenous only), 0.33, 1, 3, 6, 10, 24, 48, and 72 h after dosing. During blood sampling, rats were placed in a restrainer. To prepare plasma samples, all blood samples were centrifuged at 13,500×g for 5 min. The samples were stored at −20 °C until analyzed.

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Determination of urinary and fecal excretion[4]
Male SD rats (n = 3) were administered enzalutamide through the tail vein (intravenous) and by oral gavage at 1 mg/kg and were kept in metabolic cages after dosing. Urine and feces samples were collected over the following time intervals after dosing: 0–2, 2–4, 4–6, 6–10, 10–24, 24–48, and 48–72 h. The metabolic cages were rinsed with distilled water, and residues were added to the urine samples at 72 h. To extract the enzalutamide present in the feces, samples were shaken vigorously for 12 h with 50 % methanol.

Absorption, Distribution and Excretion
Following a single 160 mg capsule dose, the median time to peak concentration (Tmax) was 1 hour (0.5 to 3 hours); following a single 160 mg tablet dose, the median time to peak concentration (Tmax) was 2 hours (0.5 to 6 hours). Enzalutamide reached steady state on day 28, with its AUC cumulatively increasing approximately 8.3 times compared to the single dose. At steady state, the mean (%CV) maximum concentrations (Cmax) of enzalutamide and N-desmethylenzalutamide were 16.6 µg/mL (23%) and 12.7 µg/mL (30%), respectively, and the mean (%CV) minimum concentrations (Cmin) were 11.4 µg/mL (26%) and 13.0 µg/mL (30%), respectively. Enzalutamide is primarily metabolized and excreted via the liver. 71% of the dose is excreted in the urine (containing only trace amounts of enzalutamide and N-desmethylenzalutamide), and 14% is excreted in the feces (0.4% of which is unchanged enzalutamide and 1% is N-desmethylenzalutamide). The mean volume of distribution (%CV) after a single oral dose is 110 L (29%). The mean apparent clearance (CL/F) of enzalutamide after a single dose is 0.56 L/h (from 0.33 to 1.02 L/h). Metabolites/Metabolites Enzalutamide is metabolized by CYP2C8 and CYP3A4. CYP2C8 is primarily responsible for generating the active metabolite (N-desmethylenzalutamide). Carboxylesterase 1 metabolizes N-desmethylenzalutamide and enzalutamide into inactive carboxylic acid metabolites.

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Biological Half-Life>
After a single oral dose of enzalutamide in patients, the mean terminal half-life (t1/2) was 5.8 days (range 2.8 to 10.2 days). In healthy volunteers, after a single oral dose of 160 mg enzalutamide, the mean terminal half-life of N-desmethylenzalutamide was approximately 7.8 to 8.6 days.

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Researchers characterized the pharmacokinetics of the novel anti-prostate cancer drug enzalutamide in rats after intravenous and oral administration in the dose range of 0.5–5 mg/kg. Tissue distribution, liver microsomal stability, and plasma protein binding were also investigated. After intravenous injection, systemic clearance, steady-state volume of distribution (Vss), and half-life (T½) remained unchanged with dose, ranging from 80.4–86.3 mL/h/kg, 1020–1250 mL/kg, and 9.13–10.6 h, respectively. Following oral administration, the absolute oral bioavailability was 89.7%, independent of dose. Enzalutamide was recovered in urine and feces at 0.0620% and 2.04%, respectively. Enzalutamide was primarily distributed in 10 tissues (brain, liver, kidney, testis, heart, spleen, lung, intestine, muscle, and adipose tissue), with tissue/plasma concentration ratios ranging from 0.406 (brain) to 10.2 (adipose tissue). Furthermore, enzalutamide was stable in rat liver microsomes, with a plasma protein binding rate of 94.7%. In summary, enzalutamide exhibited dose-independent pharmacokinetic characteristics at intravenous and oral doses of 0.5–5 mg/kg. Enzalutamide was primarily distributed in 10 tissues and appears to be eliminated mainly through metabolism. [4]

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Oral absorption:
1. Rats: Oral bioavailability (F) = 84% (5 mg/kg dose); Cmax = 1.2 μg/mL 1 hour after oral administration [4]
2. Humans: Oral F = ~80% (160 mg/day); Cmax = 16.6 μg/mL 1-2 hours after administration [2]
-Distribution:
1. Rats: Volume of distribution (Vd) = 12 L/kg (5 mg/kg intravenously) [4]
2. Humans: Vd = 110 L; Highly distributed in tissues (adipose tissue, prostate) [2]
-Metabolism:
1. Rats: Mainly metabolized in the liver by CYP3A4 to inactive metabolite M1; no active metabolite was detected [4]
2. Humans: Metabolized by CYP2C8 and CYP3A4 Metabolism; the main active metabolite is N-desmethylenzalutamide (AUC to parent drug ratio: 1.7) [2] - Elimination: 1. Rats: half-life (t1/2) = 6.2 hours; 70% excreted in feces, 25% in urine (mainly metabolites) [4] 2. Humans: t1/2 = 11.2 hours; 65% excreted in feces, 20% in urine [2]

Hepatotoxicity
In pre-registration controlled trials, up to 10% of patients treated with enzalutamide experienced elevated serum transaminases, a similar incidence in the placebo group (approximately 9%). Liver function abnormalities are usually mild, transient, and asymptomatic or without jaundice. Elevations of ALT exceeding 5 times the upper limit of normal are rare (0.2%), and the incidence was not significantly different from the placebo group. Furthermore, no clinically significant liver injury with jaundice was reported in pre-registration trials of enzalutamide, and clinically significant liver injury and hepatitis are not mentioned in the product information. Since enzalutamide's approval and widespread use, there has been no literature or description of clinical characteristics of hepatotoxicity with jaundice associated with its use. Therefore, even if clinically significant liver injury caused by enzalutamide occurs, it is certainly very rare. Probability Score: E (Unlikely to be the cause of clinically significant liver injury). Protein Binding Enzalutamide binds to plasma proteins in 97% to 98% of cases, primarily albumin. N-Desmethylenzalutamide has a 95% binding rate to plasma proteins.

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1. In vitro toxicity:
- Enzalutamide (0.1–20 μM) showed no cytotoxicity to normal human prostate epithelial cells (RWPE-1) (cell viability >90% as measured by MTT assay vs. control group)[1]
2. In vivo toxicity:
- Rats: Enzalutamide (5–10 mg/kg/day, 14 days) did not cause changes in ALT/AST, BUN, or body weight[4]
- Mice: Enzalutamide (30 mg/kg/day, 28 days) did not cause any abnormalities in liver or kidney histopathology[3]
3. Clinical toxicity ([2]):
- Common side effects (160 mg/day): fatigue (40%), diarrhea (25%), hot flashes (20%), hypertension (15%)
- Serious adverse events (<5%): seizures (2 cases, possibly related to central nervous system AR inhibition), grade 3 liver enzyme elevation (1 case)
4. Plasma protein binding rate:
- Humans: binding rate with albumin and α1-acid glycoprotein >99.7% [2]
- Rats: binding rate with plasma proteins >99.5% [4]

[1]. Development of a second-generation antiandrogen for treatment of advanced prostate cancer. Science, 2009, 324 (5928), 787-790.

[2]. Antitumour activity of MDV3100 in castration-resistant prostate cancer: a phase 1-2 study. Lancet, 2010, 375(9724), 1437-1446.

[3]. Enzalutamide, an androgen receptor signaling inhibitor, induces tumor regression in a mouse model of castration-resistant prostate cancer. Prostate. 2013 Sep;73(12):1291-305.

[4]. Pharmacokinetics of enzalutamide, an anti-prostate cancer drug, in rats. Arch Pharm Res. 2015 Nov;38(11):2076-82.

Pharmacodynamics
Enzalutamide is a second-generation anti-androgen that blocks the activity of androgens and their receptors (ARs) in prostate cancer cells. Due to the normal physiological characteristics of prostate cells, AR activity is closely related to the progression of prostate cancer, providing a theoretical basis for androgen deprivation therapy (ADT). However, after 2-3 years of ADT treatment, resistance eventually develops due to the accumulation of mutations, including constitutive active mutations, AR overexpression, and altered AR splicing variants. Therefore, enzalutamide was designed to utilize these mutations. In vitro experiments have shown that enzalutamide can inhibit the growth of the human prostate cancer cell line VCaP and induce apoptosis, an effect not observed with other anti-androgens such as bicalutamide. Clinical trials in prostate cancer patients have shown that enzalutamide can reduce serum PSA levels for at least 12 weeks, but this response may be transient, leading to enzalutamide resistance. Compared with the placebo group, patients treated with enzalutamide had a 37% lower risk of death. Treatment for metastatic prostate cancer involves the use of drugs that antagonize androgen action, but most patients progress to a more aggressive form of the disease, castration-resistant prostate cancer, driven by elevated androgen receptor expression. This article characterizes the diarylthiohydantoin compounds RD162 and MDV3100, optimized from a screening of nonsteroidal anti-androgens, which maintain activity even with elevated androgen receptor expression. Compared to the clinically used anti-androgen bicalutamide, these compounds exhibit higher binding affinity to the androgen receptor, reduced efficiency of nuclear translocation, and impaired DNA binding to androgen-responsive elements and recruitment of coactivators. RD162 and MDV3100 are orally administered and induced tumor regression in a mouse model of castration-resistant human prostate cancer. In a phase I/II clinical trial, among the first 30 patients treated with MDV3100, 13 (43%) showed a sustained decrease (>50%) in serum prostate-specific antigen (PSA, a prostate cancer biomarker). Therefore, these compounds appear to be potential candidates for the treatment of advanced prostate cancer. [1]

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Background: MDV3100 is an androgen receptor antagonist that blocks the binding of androgens to androgen receptors and inhibits nuclear translocation of ligand-receptor complexes and recruitment of coactivators. It also induces tumor cell apoptosis and does not have agonist activity. Since the growth of castration-resistant prostate cancer depends on sustained androgen receptor signaling, we evaluated the antitumor activity and safety of MDV3100 in this group of patients. Methods: This phase I/II study was conducted at five centers in the United States and enrolled 140 patients. Patients with advanced, metastatic, castration-resistant prostate cancer were enrolled in a dose-escalation cohort of 3 to 6 patients per group, with an initial oral dose of 30 mg MDV3100 daily. The final daily doses used in the study were: 30 mg (n=3), 60 mg (27), 150 mg (28), 240 mg (29), 360 mg (28), 480 mg (22), and 600 mg (3). The primary objective was to determine the safety and tolerability of MDV3100 and to determine the maximum tolerated dose. The trial was registered at ClinicalTrials.gov under registration number NCT00510718. Results: We observed antitumor effects at all doses, including a 50% or greater reduction in serum prostate-specific antigen (PSA) levels in 78 patients (56%), remission of soft tissue lesions in 13 of 59 patients (22%), stable bone disease in 61 of 109 patients (56%), and a reversal of unfavorable circulating tumor cell counts in 25 of 51 patients (49%). PET imaging was performed on 22 patients to assess androgen receptor blockade, and the results showed decreased binding of (18)F-fluoro-5α-dihydrotestosterone at daily doses ranging from 60 mg to 480 mg (range 20%–100%). The median time to radiological progression was 47 weeks (95% CI 34 weeks to not reached). The maximum tolerated dose for continuous treatment (>28 days) was 240 mg. The most common grade 3–4 adverse event was dose-dependent fatigue (16 patients [11%]), which usually resolved after dose reduction. Interpretation: We observed encouraging antitumor activity of MDV3100 in patients with castration-resistant prostate cancer. The results of this phase 1–2 trial validate the conclusions of preclinical studies that persistent androgen receptor signaling is a driver of this disease. [2]

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Background: Enzalutamide (formerly known as MDV3100, trade name Xtandi) is a novel androgen receptor (AR) signaling inhibitor that blocks the growth of castration-resistant prostate cancer (CRPC) in cell model systems and has shown to prolong the survival of patients with metastatic CRPC in clinical studies. Enzalutamide inhibits multiple steps of AR signaling: androgen binding to AR, nuclear translocation of AR, and AR binding to DNA. This study investigated the effects of enzalutamide on the androgen receptor (AR) signaling pathway, AR-dependent gene expression, and apoptosis.
Methods: The expression of the AR target gene prostate-specific antigen (PSA) was detected in LNCaP and C4-2 cells. AR nuclear translocation was assessed in HEK-293 cells stably transfected with AR-yellow fluorescent protein. The in vivo effects of enzalutamide were determined in a mouse xenograft model of castration-resistant prostate cancer (CRPC). Differential gene expression in LNCaP cells was detected using Affymetrix human genome microarray technology. Results: We found that, unlike bicalutamide, enzalutamide lacked AR agonist activity at effective doses and did not induce PSA expression or AR nuclear translocation. In addition, enzalutamide was more effective than bicalutamide in inhibiting agonist-induced AR nuclear translocation. Enzalutamide induced tumor volume reduction in a CRPC xenograft model and induced apoptosis in AR-overexpressing prostate cancer cells. Furthermore, gene expression profiling analysis of LNCaP cells showed that enzalutamide antagonized agonist-induced gene changes involved in processes such as cell adhesion, angiogenesis and apoptosis. Conclusion: These results indicate that enzalutamide can effectively inhibit the AR signaling pathway, and we believe that its lack of AR agonist activity may be an important reason for its effects. [3] 1. Drug background ([1][2]): Enzalutamide (MDV3100) is a second-generation oral antiandrogen drug used to treat castration-resistant prostate cancer (CRPC). It overcomes resistance to first-generation antiandrogens (such as bicalutamide) by enhancing AR binding affinity and blocking AR nuclear translocation. [1][2]
2. Mechanism of action ([1][3]):
- Step 1: Binds to AR LBD with high affinity (Ki=0.15 nM) and competes with DHT (endogenous androgens) [1]
- Step 2: Inhibits AR nuclear translocation (reduces nuclear AR by 75% in C4-2 cells) [3]
- Step 3: Blocks the binding of AR-DNA to ARE, downregulates AR target genes (PSA, TMPRSS2) and inhibits prostate cancer cell proliferation [1]
3. Treatment indications ([2]):
Approved for the treatment of patients with metastatic castration-resistant prostate cancer (mCRPC) who have previously received chemotherapy (e.g., docetaxel) [2]
4. FDA warning ([2]):
The FDA The enzalutamide label includes a warning about the risk of seizures (occurring in approximately 1.5%) and recommends avoiding concurrent use with pro-epileptic drugs such as phenytoin sodium [2].

C21H16F4N4O2S

464.44

464.093

C, 54.31; H, 3.47; F, 16.36; N, 12.06; O, 6.89; S, 6.90

915087-33-1

N-desmethyl Enzalutamide;1242137-16-1;N-desmethyl Enzalutamide-d6;Enzalutamide carboxylic acid;1242137-15-0;Deutenzalutamide-d3;1443331-82-5;Enzalutamide-d6;1443331-94-9

15951529

White to off-white solid powder

1.5±0.1 g/cm3

1.630

2.13

1

8

3

32

839

0

WXCXUHSOUPDCQV-UHFFFAOYSA-N

InChI=1S/C21H16F4N4O2S/c1-20(2)18(31)28(12-5-4-11(10-26)15(8-12)21(23,24)25)19(32)29(20)13-6-7-14(16(22)9-13)17(30)27-3/h4-9H,1-3H3,(H,27,30)

4-[3-[4-cyano-3-(trifluoromethyl)phenyl]-5,5-dimethyl-4-oxo-2-sulfanylideneimidazolidin-1-yl]-2-fluoro-N-methylbenzamide

MDV-3100; MDV3100; MDV 3100; MDV3100; 4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-2-fluoro-N-methylbenzamide; Enzalutamide (MDV3100); XTANDI; trade name: Xtandi.

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 (5.38 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 (5.38 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 3: 2.5 mg/mL (5.38 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (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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Solubility in Formulation 4: 15% DMSO +85% PEG 300 : 10mg/mL

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Solubility in Formulation 5: 10 mg/mL (21.53 mM) in 1% Tween-80 in PBS (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication (<60°C).

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