01 M Tris�CHCl, www.selleckchem.com/products/mek162.html 0.15 M sodium chloride, pH 7.4, containing 0.1% gelatin), were incubated in a test tube at 37 ��C for 1 hr. Normal rabbit serum (0.1 ml of a 1/25 dilution) was added to each tube to control nonspecific binding. Goat antirabbit immunoglobulin (0.1 ml) is then added, and the mixture was incubated at 4 ��C overnight. The precipitate was collected by centrifugation at 1,000 �� g for 30 min at 5 ��C, the supernatant was decanted, and the walls of the tubes were wiped dry. To count [3H]nicotine or [3H]cotinine, the precipitant was dissolved in 0.1-ml 0.1 N sodium hydroxide before adding 2.5 ml of liquid scintillation fluid (Bioflour from Packard Instrument Company, Meriden, CT). Radioactivity was measured in an LKB-Wallac liquid scintillation counter (LKB Instrument Inc.

, Gaithersburg, MD). Quantification of nicotine or cotinine in the experimental plasma specimens was accomplished by using their percent inhibition of immune binding to the respective antibody (i.e., antinicotine or anticotinine) versus the logarithmically transformed known concentrations of unlabeled analyte added. Cross-reactivity of the nicotine and cotinine antibodies with other nicotine metabolites was less than 5%. The detectable concentration ranges for nicotine and cotinine in rat plasma were 1.25�C62.5 and 0.5�C50 ng/ml, respectively. The slope and intercept values show little interday variations (coefficients of variation of less than 6%). Data Interpretation All pharmacokinetic analyses were made using classical techniques and the microcomputer-based program WinNonlin by Pharsight, Inc.

The nicotine and cotinine plasma concentration�Ctime data were interpreted by noncompartmental methods. The highest observed plasma concentration and its time of occurrence were defined as Cmax and Tmax. The rate constant governing elimination of nicotine from the body (��) or elimination of cotinine from the body (k) was determined from the least-squares slope of the terminal linear segment of a semi-logarithmic plot of plasma nicotine or cotinine concentration versus time. The biological half-life of nicotine or cotinine was calculated as 0.693/�� or 0.693/k. The total area under the plasma concentration�Ctime curve (AUC0�C4 or AUC��) and the area under the first moment of the plasma concentration�Ctime Dacomitinib curve (AUMC) were estimated by the trapezoidal rule with extrapolation of the terminal portion to infinity for the single-cigarette smoke inhalation studies. The mean residence time (MRT) was calculated from AUMC/AUC. Differences between any two mean pharmacokinetic parameter values were evaluated statistically by the Student��s t test for unpaired (unrelated) or paired (related) samples at p = .05.