Ketoconazole (R-41400), a CYP3A4 inhibitor, is an imidazole anti-fungal agent.

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

Intraperitoneal administration of Ketoconazole (25 mg/kg) in rats significantly reduced plasma corticosterone levels and decreased self-administration of low doses of cocaine. Rats treated with Ketoconazole exhibited enhanced bioavailability of digoxin, increasing from 0.68 to 0.84, with the mean absorption time decreasing from 1.1 h to 0.3 h. Moreover, the oral area under the curve (AUC) for digoxin increased from 63 mg·h/L to 411 mg·h/L, while the intravenous AUC also rose from 93 mg·h/L to 486 mg·h/L.

In HT29-S-B6 colorectal cancer cells, Ketoconazole decreased cell proliferation and [3H]thymidine uptake in a dose-dependent manner, with an IC50 of 2.5 mM. Ketoconazole also inhibited [3H]thymidine uptake in both Evsa-T and MDA-MB-231 cell lines, with respective IC50 values of 2 μM and 13 μM. Within 24 hours, Ketoconazole induced a dose-dependent reduction in the S-phase cell population (from 17% to 3%) and a corresponding increase in the Go-G1 phase cell percentage (from 64% to 80%) in HT29-S-B6 cells. By competitively binding with [3H]Dexamethasone, Ketoconazole inhibited fibroblast glucocorticoid receptors, with an IC50 of 0.3 mM. Several Aspergillus species were sensitive to Ketoconazole, with a minimum inhibitory concentration of 0.03 μg/mL.

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)