Enzalutamide

In Vitro

Aliquot the stock solution and store it at -80°C (up to 2 years) or -20°C (up to 1 year); avoid repeated freeze-thaw cycles.

In Vivo

Choose the formulation that suits the animal model and route of administration; percentages are volume ratios of the final working solution. Start from a clear DMSO stock (see In Vitro above), add the co-solvents one at a time in the order listed, mixing after each addition, and prepare the working solution fresh on the day of dosing. If precipitation or phase separation occurs, gentle warming or sonication can help.

Direct preparation of the working solution

These formulations are prepared directly, without a DMSO stock; use them promptly after preparation.

Data provided by the manufacturer.

In Vitro

In a competition assay with 16β-[¹⁸F]fluoro-5α-DHT (18-FDHT) in castration-resistant, AR-overexpressing LNCaP/AR cells, Enzalutamide (MDV3100) binds AR with greater affinity than ICI 176334. Enzalutamide shows no agonism in LNCaP/AR prostate cells. In parental LNCaP cells, Enzalutamide blocks the induction of both prostate-specific antigen (PSA) and transmembrane serine protease 2 (TMPRSS2) when combined with the synthetic androgen R1881. Enzalutamide suppresses transcriptional activity of the mutant AR protein W741C (mutation of Trp741 to Cys)^[1]. Nuclear translocation and co-activator recruitment of the ligand-receptor complex are also prevented by Enzalutamide^[2].

In Vivo

In castrate male mice bearing LNCaP/AR xenografts, Enzalutamide (MDV3100) at 10 mg/kg induces marked tumor regression^[1]. Enzalutamide displays dose-independent pharmacokinetics across intravenous and oral doses of 0.5-5 mg/kg^[4].

Data provided by the manufacturer. Numbered citations refer to the Literature Cited list in the product description.

Cell Assay^[1]

Culture LNCaP cells (10⁷ cells/condition) for 22 days in RPMI media containing 5% charcoalstripped serum, then treat for 8 hours with DMSO or 1 nM R1881 together with one antiandrogen: DMSO; 1 μM ICI 176334 or 10 μM ICI 176334; 1 μM RD162 or 10 μM RD162; 1 μM MDV3100 or 10 μM MDV3100. Harvest an aliquot of cells for qRT-PCR of PSA and TMPRSS2 mRNA. Cross-link the remaining cells with 1% paraformaldehyde for 10 minutes, then add glycine and centrifuge the samples (4°C, 4000 rpm, 5 minutes) to stop further crosslinking. Perform chromatin immunoprecipitation with a chromatin immunoprecipitation assay kit. Amplify the immunoprecipitated DNA by real-time PCR. Use these primers: PSA enhancer forward-ATGTTCACATTAGTACACCTTGCC, reverse-TCTCAGATCCAGGCTTGCTTACTGTC; TMPRSS2 enhancer forward-TGGTCCTGGATGATAAAAAAAGTTT, reverse-GACATACGCCCCACAACAGA^[1].

Animal Administration^[3][4]

Mice^[3] After a 5-day acclimation period, castrate 5- to 9-week-old male CB17SCID mice and allow 5 additional days of recovery before tumor cell inoculation. Generate a xenograft model of human prostate cancer using LNCaP-AR-Lux cells (LNCaP cells that express exogenous AR together with the AR-dependent reporter construct ARR2-Pb-Luc). Before implantation, treat LNCaP-AR-Lux cells with trypsin-EDTA, wash with complete medium, collect, and resuspend at 20×10⁶ cells/mL. Dilute the cell suspensions with Matrigel to 2×10⁶ cells/0.2 mL and inject subcutaneously in the suprascapular region. Monitor tumor growth until the volume reaches 100 mm³, when treatment begins (80 days). The observed tumor take rate with LNCaP-AR-Lux cells is between 70% and 80%. Measure body weight and tumor volumes (width²×length/2) two to three times per week with a digital caliper, and determine the average tumor volumes. Dilute test drugs in Tween 80:PEG 400 and store at 4°C until administration by oral gavage. Treat each group of mice (n=7) once daily for 28 consecutive days with Enzalutamide at 1, 10, or 50 mg/kg, a vehicle control, or ICI 176334 at 50 mg/kg. Euthanize the animals when the treatment period ends or tumor volume exceeds 1,000 mm³, and collect blood and tissue samples for analysis. Rats^[4] Administer Enzalutamide to male SD rats (n=3) through the tail vein (intravenous) and by oral gavage at 1 mg/kg, and keep the rats in metabolic cages after dosing. Collect urine and feces samples at 0-2, 2-4, 4-6, 6-10, 10-24, 24-48, and 48-72 h after dosing. Rinse the metabolic cages with distilled water and add the residues to the urine samples at 72 h. To extract Enzalutamide from the feces, shake the samples vigorously for 12 h with 50 % methanol.

Data provided by the manufacturer. Numbered citations refer to the Literature Cited list in the product description.