Ketoconazole

Ketoconazole (R-41400) is an imidazole antifungal agent with broad-spectrum antifungal activity that primarily acts by inhibiting the biosynthesis of ergosterol in the fungal cell membrane. Ketoconazole inhibits the fungal cytochrome P450-dependent enzyme lanosterol 14α-demethylase (CYP51), thereby blocking the conversion of lanosterol to ergosterol. This leads to damage to the cell membrane structure and permeability, consequently inhibiting fungal growth and producing an antifungal effect. In addition, ketoconazole is a non-selective cytochrome P450 (CYP) inhibitor, specifically inhibiting drug-metabolizing enzymes such as human CYP3A4. In terms of endocrinology, ketoconazole also inhibits various enzymes involved in steroid synthesis (CYP17A1, CYP11A1).

Cyclosporine oxidase:0.19 mM, Testosterone 6β-hydroxylase:0.22 mM

Methods: Human liver microsomes were incubated at 37°C with ketoconazole (0.3, 1, 2, 3, 5, 10 μM) and CYP3A-specific substrates testosterone and midazolam. High-performance liquid chromatography (HPLC) was used to quantify the formation of metabolites (6β-hydroxy testosterone and 1'-hydroxy midazolam).
Results: Ketoconazole inhibited CYP3A activity in a dose-dependent manner at all concentrations tested, reducing activity to below 3% (for testosterone) at 10 μM. [1]
Methods: U87 glioma cells and patient-derived glioblastoma stem cells (GSCs) were treated with ketoconazole at concentrations ranging from 0.1 to 100 μM for 48–72 hours. Cell proliferation and apoptosis were assessed via Annexin V/PI staining.
Results: Ketoconazole treatment reduced cell proliferation and increased apoptosis rates. [2]

Methods: U87-luc cells or patient-derived GSC were implanted in situ into the brains of immunodeficient mice. After tumor formation, mice were randomly assigned to treatment groups. Daily intraperitoneal injections of Ketoconazole (50 mg/kg) or solvent control were administered for 4 consecutive weeks. Tumor growth was monitored via bioluminescence imaging, and mouse survival was recorded.
Results: Tumor growth was significantly suppressed in the ketoconazole group, with markedly prolonged mouse survival. Histological analysis revealed reduced tumor cell proliferation, increased apoptosis, and decreased metabolic activity within tumors. [2]
Methods: Wild-type mice received intraperitoneal injections of ketoconazole (50 mg/kg), followed approximately 30 minutes later by intravenous administration of a radiolabeled tracer ([¹¹C]lopipridil or [¹¹C]dLop). Mice were euthanized 30 minutes after tracer injection, and blood and whole brain samples were collected.
Results: Ketoconazole (50 mg/kg, i.p.) partially inhibited the N-demethylation of [¹¹C]lopipridil in vivo, elevated its plasma concentration, and reduced the entry of polar metabolites into the brain. [3]

Whole Cell [3H]R1881 Binding Assay: Fibroblasts are grown to confluence in five or six 150 cm2 tissue culture flasks for routine assay. This usually requires 4-6 weeks from the time of the initial seeding of the cell line. All studies are performed between passages 3-20. Two days before assay, the medium is changed to one lacking fetal calf serum. This is repeated again 24 hours before assay. Competition assays are performed with 0.5-1.0 nM [3H]R1881 and increasing amounts of the nonradioactive compounds. Binding to low affinity sites is determined in the presence of 5 × 10-7 M R1881 and is subtracted from whole cell binding of [3H]R 1881 obtained in the absence of any inhibitor to assess binding to 5 high affinity site

HT29-S-B6 cells (5×105) are plated in 35-mm Petri dishes. The next day, the medium is changed and effectors are added in a small volume (10-20 μL). The incubation medium is renewed every day during the experiments. The same triplicate dishes are used for cell counts, [3H]thymidine incorporation, and flow cytometry. [3H]Thymidine (0.5 μCi) is allowed to incorporate for 24 hours; at the end of incubation, cells are rinsed with 1 mL of medium, detached with 1 mL of trypsin-EDTA, and diluted (1:3) with the culture medium. An aliquot (0.5-1 mL) is used for cell count with a Coulter Counter.(Only for Reference)

DMSO: 25 mg/mL (47.04 mM), Sonication and heating are recommended.

10% DMSO+40% PEG300+5% Tween 80+45% Saline: 0.53 mg/mL (1 mM), Solution.