A Sensitive Bioassay for Measuring Blood Levels of 12-O-Tetradecanoylphorbol-13-acetate (TPA) in Patients: Preliminary Pharmacokinetic Studies

12-O-Tetradecanoylphorbol-13-acetate (TPA) is a potent stimulator of differentiation in myelocytic leukemia cells, and it has been shown to have activity in patients with acute myelocytic leukemia. Because attempts to develop a suitable mass spectrometry assay for TPA were unsuccessful (because of the lack of sufficient sensitivity), we developed a novel and highly sensitive blood level bioassay for TPA that measures ethyl acetate-extractable differentiating activity in blood. Differentiating activity in ethyl acetate extracts of blood was measured in HL-60 cells by measuring the formation of adherent cells. The sensitivity of the assay was ∼0.1 ng TPA/ml blood. The assay for TPA has a high degree of specificity and does not measure deesterifed potential metabolites (phorbol, phorbol-13-acetate, or phorbol-12-myristate), and the presence of GM-CSF, G-CSF, interferon-α, or interferon-γ does not interfere with the assay. Blood levels of TPA as measured by the bioassay immediately after an IV infusion of TPA (0.125 mg/m²; ∼0.25 mg per patient) and 1 and 3 h later were 1.75 ± 0.55, 0.93 ± 0.54, and 0.69 ± 0.42 ng/ml, respectively (mean ± SD from eight infusions in five patients). Terminal half-lives were determined in a few patients where TPA blood levels were measured at multiple time intervals after the TPA infusion. In these patients, the terminal half-life was 11.1 ± 3.9 h (from five infusions in four patients). To the best of our knowledge, this is the first analytical method for the measurement of TPA.