Effects of TRAM-34 on rat CYP Activity.

<p>Recombinant enzyme CYP2C6 (A), CYP2C11 (B), CYP1A2r (C) and CYP2B1 (D), substrate and varying concentrations of TRAM-34 were incubated in the presence of 50 mM potassium phosphate buffer and regenerating system at 37°C according to the methods described. Percent control enzyme activity (ordinate) is plotted versus the log of inhibitor concentration (abscissa). All TRAM-34 and clotrimazole data points (A and B) represent the mean (±SEM) of 3 experiments performed in triplicates. Other clotrimazole data represent the mean of duplicates (C) or triplicates (D) from a single experiment. TRAM-34 IC₅₀ values were determined by non-linear regression and are shown in parentheses. Control enzyme activities were (mean ± SEM, n = 3 experiments each) 1.65±0.34 (<b>A</b>), 0.14±0.02 (<b>B</b>), 0.68±0.11 (<b>C</b>) and 6.13±0.7 (<b>D</b>) min^–1. In this and subsequent figures, error bars represent SEM of measurements, but, due to the small variability, are not always visible.</p

Concentration-dependent inhibition and activation of CYP3A4 by TRAM-34 with two substrates.

<p>Recombinant enzyme CYP3A4, substrates DBF (A) or BFC (B) and varying concentrations of TRAM-34 were incubated in the presence of 50 mM potassium phosphate buffer and regenerating system at 37°C according to the methods described. Pmol of product (ordinate) is plotted versus the log of inhibitor concentration (abscissa) for the incubation times specified in parenthesis. All TRAM-34 data points represent the mean ±SEM of 3 experiments performed in triplicate. Data from ketoconazole represent the mean ±SEM of triplicates from a single experiment. The TRAM-34 IC₅₀ value was determined by non-linear regression and are shown in brackets (A). Control data points (i.e. no inhibitor, C on abscissa) represent the mean ±SEM from 3 experiments.</p

Effects of TRAM-34 on human CYP Activity.

<p>Recombinant enzyme CYP2C19 (A), CYP19A1h (B), CYP1A2h (C) and CYP2B6 (D), substrate and varying concentrations of TRAM-34 were incubated in the presence of 50 mM potassium phosphate buffer and regenerating system at 37°C according to the methods described. Percent control enzyme activity (ordinate) is plotted versus the log of inhibitor concentration (abscissa). All TRAM-34 (<b>A–D</b>) and fluvoxamine (<b>C</b>) data points represent the mean (±SEM) of 3 experiments performed in triplicates. Data from the other inhibitors (A, B and D) represent the mean (±SEM) of triplicates from a single experiment. TRAM-34 IC₅₀ values were determined by non-linear regression and are shown in parentheses. Control enzyme activities were (mean ± SEM, n = 3 experiments) 0.30±0.03 (<b>A</b>), 0.13±0.003 (<b>B</b>), 4.26±0.09 (<b>C</b>) and 3.81±0.32 (<b>D</b>) min⁻¹.</p

Conditions for CYP Assays.

<p>Conditions used for the present CYP assays are summarized. All assays used fluorescence plate reader methods except where noted otherwise.</p>a<p>See text for regenerating system compositions.</p>b<p>LC-MS/MS methods used for this assay only.</p