Chemicals and reagents
Imatinib mesylate was supplied by Novartis (East Hanover, NJ). Tariquidar was supplied by Dr. Susan Bates (NCI, Bethesda, MD). Glucose, harmine, absolute ethanol and ammonium acetate were purchased from Sigma-Aldrich (St. Louis, MO). Formic acid (98%) was obtained from Fluka (through Sigma-Aldrich). Methanol (J.T. Baker, Phillipsburg, NJ) was of HPLC grade. Deionized water was generated with a Hydro-Reverse Osmosis system (Durham, NC) connected to a Milli-Q UV Plus purifying system (Billerica, MA). Blank mouse plasma was purchased from Innovative Research (Southfield, MI).
Sample Preparation
Unknown and quality control (QC) plasma samples were thawed at room temperature, vortex mixed for 20 seconds, and 100 μL were transferred to a polypropylene centrifuge tube. For analysis of unknown tissue samples, approximately 100 mg of tissue were accurately weighed and water added (5 μL per mg). After vortex-mixing, samples were homogenized using a PowerGen 125, while kept on ice. One hundred μL of homogenate was transferred to a clean polypropylene centrifuge tube for further processing. To each tube, including calibrators (10, 25, 50, 100, 500 and 1000 ng/mL) and QC samples (30, 450, 800 and 18,000 ng/mL), 250 μL of methanol (containing 25 ng/mL of internal standard, harmine) was added. All tubes were capped, vortex-mixed for 5 min and then centrifuged for 5 min at 18,000 × g. Following centrifugation, the supernatant was transferred to a vial for injection. Either 5 or 10 μL of the supernatant was injected for tissue or plasma samples, respectively. Calibration curves and QC samples were prepared in both brain and liver, for tissue sample analysis. The working ranges for liver and brain were 0.125–100 and 0.125–25 ng/mL, respectively.
Equipment
High performance liquid chromatography was carried out on an Agilent 1100 system (Agilent Technology, Palo Alto, CA), coupled with a single-quadrupole mass spectrometer, utilizing electrospray ionization in positive mode. Samples were cooled to 4°C in a thermostated autosampler and the column compartment, containing a Waters SymmetryShield RP8 column (2.1 × 50 mm, 3.5 μm), was maintained at 35°C. Samples were eluted using a gradient mobile phase, comprised of 10 mM ammonium acetate with 0.1% formic acid and methanol, running at a flow rate of 0.35 mL/min for 10 min, including re-equilibration. Mass spectrometric conditions were as follows: fragmentor, 150 V; gain, 2; drying gas flow, 10 L/min; drying gas temperature, 300°C; nebulizer pressure, 40 psi; and capillary voltage, 1500 V. Selected-ion monitoring was accomplished at m/z 494.2 for imatinib and m/z 213.1 for the internal standard. The chromatographic data were acquired and analyzed using the Chemstation software package (Agilent).
Validation procedures
Calculation of accuracy and precision was carried out according to procedures reported in detail previously [17]. Calibration samples were prepared fresh each day in the relevant matrix and frozen QC samples were defrosted and analyzed. A 1/x2 weighting scheme was employed in the generation of standard curves to account for concentration dependent variance. Detector response for plasma was found to be linear in the imatinib concentration range of 10–1000 ng/mL. Plasma accuracy and precision were evaluated with QC samples. Overall, the assay was found to be accurate (deviation of less than 10% for QCs) and precise (within run precision <10%, between run precision <12.6%) for plasma, liver, and brain.
Animals
All experiments were performed on six-week old, male, Balb/C mice obtained from Charles River Laboratories (Wilmington, MA). The mice weighed approximately 15 to 20 g at the time of study. All mice were allowed unlimited access to water and rodent chow prior to, and during the experiment. Blank mouse liver and brain samples were harvested from surplus mice following euthanasia. NCI-Frederick is accredited by AAALAC International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the "Guide for Care and Use of Laboratory Animals" (National Research Council; 1996; National Academy Press; Washington, DC). The study design and protocol were approved by the NCI Animal Care and Use Committee (Bethesda, MD).
Experimental Design
Imatinib was dissolved in sterile water to make a 10 mg/mL dosing solution. Tariquidar was prepared as a 2 mg/mL solution in water with 5% glucose. Mice received either 10 mg/kg tariquidar or the vehicle (5 mL per kg weight) [15] 30 minutes prior to 50 mg/kg of imatinib [18]. All compounds were administered via oral gavage. At each time point, three mice in each treatment group were anesthetized with isoflurane, and bled via cardiac puncture into a tube containing sodium heparin as an anticoagulant. Blood samples were centrifuged at 18,000 × g for 5 minutes at 4°C, the plasma layer transferred to a cryovial and frozen. Following euthanasia by cervical dislocation, brain and liver tissues were excised and snap-frozen. All samples were stored at -80°C until the time of analysis.
Statistical and pharmacokinetic analysis
Concentration-time data were evaluated using a non-compartmental approach, with WinNonlin 5.0 (Pharsight, Mountain View, CA), using the mean concentration (n = 3) at each time point. The peak plasma concentration (Cmax) and the time to peak plasma concentration (Tmax) are reported as observed values. The area under the curve (AUC) was calculated using the linear trapezoidal method from time zero to the time of the last sample with measurable drug concentration. To allow for direct comparison between the two groups and characterization of the terminal phase for the imatinib alone arm, the 24-hour plasma and liver samples, along with the 4-hour brain samples were estimated at LLQ/2, as drug was detectable, but measured concentrations were below the limit of quantitation. Bailer's method was employed to assess the variance, allowing for comparison of exposure between the two dose groups. The significance of the difference in AUC was evaluated by a Z-test. Brain concentrations were corrected for drug in the brain vascular space, by subtracting 1.4% of the plasma concentration from the measured brain concentration for each animal [5]. Brain-to-plasma concentration ratios were calculated for each animal at the 2-hour time point, and the groups compared using a t-test. All statistical tests were performed in Microsoft Excel 2004 (Redmond, WA). P-values < 0.05 were considered significant.