nonlinear regression analysis graphpad prism 9.0 software  (GraphPad Software Inc)

Journal: Chemical research in toxicology

Article Title: Cytochromes P450 2C8 and 3A Catalyze the Metabolic Activation of the Tyrosine Kinase Inhibitor Masitinib

doi: 10.1021/acs.chemrestox.2c00057

Figure Lengend Snippet: Effect of time-dependent P450 inhibitors on masitinib metabolite formation: (A) M485, (B) M515c, (C) MCN510, (D) MCN524, (E) MCN526, and (F) MCN538. Masitinib (2 μM) was incubated with pooled HLM (0.1 mg/mL) and KCN (1 mM) following preincubation in the presence or absence of time-dependent P450-selective chemical inhibitors. The following chemical inhibitors were used to block the respective P450: gemfibrozil O-β-glucuronide, Gem-O-Gluc (64 μM, P450 2C8, 30 min preincubation), furafylline (25 μM, P450 1A2, 10 min preincubation), ketoconazole (1 μM, P450 3A), and CYP3cide (2 μM, P450 3A4, 5 min preincubation). Control incubations were carried out with the appropriate vehicle solvent control in the absence of an inhibitor. Relative levels of metabolite formed were measured by LC–MS/MS. Percent (%) metabolite formation was based on comparison to vehicle control. The results are from two independent experiments performed in triplicate (n = 3 per experiment). The bar shown is the grand mean, and data points are the individual values from each replicate. Outliers were removed based on Grubbs’ outlier test (α = 0.05) using GraphPad Prism 9.0 software. Comparisons of inhibitor vs vehicle control were performed by the unpaired two-tailed t test (GraphPad Prism 9.0 Software). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: Data were fit to the following equation using nonlinear regression analysis with GraphPad Prism 9.0 software to estimate the apparent K I and k inact .

Techniques: Incubation, Blocking Assay, Liquid Chromatography with Mass Spectroscopy, Software, Two Tailed Test

Journal: Chemical research in toxicology

Article Title: Cytochromes P450 2C8 and 3A Catalyze the Metabolic Activation of the Tyrosine Kinase Inhibitor Masitinib

doi: 10.1021/acs.chemrestox.2c00057

Figure Lengend Snippet: Kinetic analysis of masitinib metabolite formation in pooled HLM: (A) M485, (B) M515c, (C) MCN510, (D) MCN524, (E) MCN526, and (F) MCN538. Masitinib (0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, and 100 μM) was incubated with pooled HLM (0.25 mg/mL) supplemented with NADPH and potassium cyanide (KCN 1 mM). Relative levels of metabolites formed were measured by LC–MS/MS. The results shown are from two independent experiments (trials 1 and 2) conducted in triplicate each (n = 3). Data points shown are from individual replicates. Data were graphed as peak area versus substrate concentration [S] (A–F) and using the Eadie–Hofstee transformation as peak area versus peak area/[S] (inset A–F). Based on comparison of kinetic model fits using the corrected Akaike’s information criterion (AICc) method in GraphPad Prism 9.0 software, the Michaelis–Menten model was selected as the preferred model for M485 (A), and substrate inhibition was the preferred model for M515c, MCN510, MCN524, MCN526, and MCN538 (B–F). Therefore, data for (A) were fit to the Michaelis–Menten model, and data for (B–F) were fit to the substrate inhibition model using nonlinear regression analysis with GraphPad Prism 9.0 software.

Article Snippet: Data were fit to the following equation using nonlinear regression analysis with GraphPad Prism 9.0 software to estimate the apparent K I and k inact .

Techniques: Incubation, Liquid Chromatography with Mass Spectroscopy, Concentration Assay, Transformation Assay, Software, Inhibition