Pharmacokinetics of 9-nitrocamptothecin (9NC) and 9- aminocamptothecin (9AC) in SCID mice bearing human colon xenografts, HT29

Abstract

5401

Introduction: 9NC is an orally administered camptothecin analogue that has completed Phase III trials in pancreatic cancer. However, limited data are available regarding tissue distribution of 9NC and its active metabolite, 9AC. We developed a sensitive and specific LC-MS/MS assay for quantitating 9NC and 9AC and used it to define the plasma and tumor pharmacokinetics of 9NC and 9AC in female SCID mice bearing HT29 human colon xenografts. Methods: 9NC was administered po at 0.44 or 1.0 mg/kg. Groups of 3 mice were euthanized at 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, 360, 420, and 1440 min after administration of 9NC or 5 min after administration of vehicle. Blood and tissues were collected, and blood was immediately centrifuged to obtain plasma. Tissues were weighed, snap frozen, and stored at -70 ^o C. 9NC and 9AC in plasma and tumor were converted to their respective lactone forms, and the concentrations of each were determined by LC-MS/MS. The LC-MS/MS assay used: camptothecin as an internal standard; either methanol (plasma) or acetonitrile (tissues) protein precipitation; a Phenomenex Luna C(18)2 (4.5 μm, 150 ×2 mm) analytical column; an isocratic mobile phase of 0.1% formic acid in acetonitrile:50 μM ammonium acetate buffer, pH 5.0 (40:60, v/v); and electropositive spray mode electron ionization with MRM detection. Concentration-versus-time data were modeled with non-compartmental methods utilizing the Lagrange function. Results: The retention times of 9AC, internal standard, and 9NC were 2.8, 4.6, and 7.9 min, respectively. The assay had a run time of 10 min, a lower limit of quantitation of 1.0 ng/ml, and was linear between 1 and 1000 ng/ml. Peak plasma 9NC and 9AC concentrations and areas under the concentration vs. time curve (AUC’s) were proportional to dose. After 0.44 and 1 mg/kg 9NC, peak plasma concentrations of 9NC were 34 and 90 ng/ml and occurred at 5 and 15 minutes, respectively. Plasma 9NC AUC was 1726 ng/ml/min at 0.44 mg/kg and 4111 ng/ml/min at 1 mg/kg. 9NC clearance was 255 ml/min/kg at 0.44 mg/kg and 243 ml/min/kg at 1mg/kg. 9NC half-lives were 86 and 35 min for doses of 9NC of 0.44 and 1 mg/kg, respectively. Peak plasma 9AC concentrations were 5 and 11 ng/ml and occurred at 5 and 15 min after 9NC doses of 0.44 and 1 mg/kg, respectively. 9AC AUC was 687 ng/ml/min at 0.44 mg/kg and 1135 ng/ml/min at 1.0 mg/kg. Peak tumor concentrations of 9NC were 5 and 25 ng/ml and occurred at 30 min after doses of 0.44 and 1 mg/kg 9NC. Tumor 9NC AUC’s were 1094 and 2016 ng/ml/min after 9NC doses of 0.44 and 1 mg/kg, respectively. Conclusion: The LC-MS/MS method developed is sensitive and robust and should prove suitable for use in clinical studies of 9NC. The 9NC data suggest that the murine pharmacokinetics are linear with dose and that tumor concentrations of 9NC and 9AC are above the concentrations required for in vitro anti-tumor activity. Support: Whitaker Foundation Grant RG-01-0311 and NCI P30 CA47904.