Limits...
TRAM-34, a putatively selective blocker of intermediate-conductance, calcium-activated potassium channels, inhibits cytochrome P450 activity.

Agarwal JJ, Zhu Y, Zhang QY, Mongin AA, Hough LB - PLoS ONE (2013)

Bottom Line: However, previously published work has only characterized the effects of TRAM-34 on a single CYP isoform.TRAM-34 also had both stimulatory and inhibitory effects on human CYP3A4 activity, depending on the substrate used.These results show that low micromolar concentrations of TRAM-34 can inhibit several rat and human CYP isoforms, and suggest caution in the use of high concentrations of this drug as a selective IKCa channel blocker.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York, United States of America.

ABSTRACT
TRAM-34, a clotrimazole analog characterized as a potent and selective inhibitor of intermediate-conductance, calcium-activated K(+) (IKCa) channels, has been used extensively in vitro and in vivo to study the biological roles of these channels. The major advantage of TRAM-34 over clotrimazole is the reported lack of inhibition of the former drug on cytochrome P450 (CYP) activity. CYPs, a large family of heme-containing oxidases, play essential roles in endogenous signaling and metabolic pathways, as well as in xenobiotic metabolism. However, previously published work has only characterized the effects of TRAM-34 on a single CYP isoform. To test the hypothesis that TRAM-34 may inhibit some CYP isoforms, the effects of this compound were presently studied on the activities of four rat and five human CYP isoforms. TRAM-34 inhibited recombinant rat CYP2B1, CYP2C6 and CYP2C11 and human CYP2B6, CYP2C19 and CYP3A4 with IC50 values ranging from 0.9 µM to 12.6 µM, but had no inhibitory effects (up to 80 µM) on recombinant rat CYP1A2, human CYP1A2, or human CYP19A1. TRAM-34 also had both stimulatory and inhibitory effects on human CYP3A4 activity, depending on the substrate used. These results show that low micromolar concentrations of TRAM-34 can inhibit several rat and human CYP isoforms, and suggest caution in the use of high concentrations of this drug as a selective IKCa channel blocker. In addition, in vivo use of TRAM-34 could lead to CYP-related drug-drug interactions.

Show MeSH

Related in: MedlinePlus

Effects of TRAM-34 on rat CYP Activity.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 (A), 0.14±0.02 (B), 0.68±0.11 (C) and 6.13±0.7 (D) min–1. In this and subsequent figures, error bars represent SEM of measurements, but, due to the small variability, are not always visible.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3646888&req=5

pone-0063028-g001: Effects of TRAM-34 on rat CYP Activity.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 (A), 0.14±0.02 (B), 0.68±0.11 (C) and 6.13±0.7 (D) min–1. In this and subsequent figures, error bars represent SEM of measurements, but, due to the small variability, are not always visible.

Mentions: TRAM-34 was tested on CYP activities from 4 rat and 5 human isoforms. Surprisingly, concentration-dependent inhibition by TRAM-34 was seen with 3 rat CYP isoforms. TRAM-34 potently inhibited CYP2C6 and CYP2B1 (IC50 = 2.9 µM and 3.0 µM respectively, Figs. 1A and 1D). The drug showed weaker inhibition on CYP2C11 with an IC50 value of 12.6 µM (Fig. 1B). Clotrimazole, used as a positive control, was a potent inhibitor of CYP2C6, CYP2B1, and CYP2C11, as expected. TRAM-34 showed no inhibition at concentrations up to 80 µM on CYP1A2r (Fig. 1C). Clotrimazole only partly inhibited CYP1A2r activity (Fig. 1C). Other inhibitors (fluvoxamine and miconazole) did not inhibit CYP1A2r activity up to 80 µM (not shown).


TRAM-34, a putatively selective blocker of intermediate-conductance, calcium-activated potassium channels, inhibits cytochrome P450 activity.

Agarwal JJ, Zhu Y, Zhang QY, Mongin AA, Hough LB - PLoS ONE (2013)

Effects of TRAM-34 on rat CYP Activity.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 (A), 0.14±0.02 (B), 0.68±0.11 (C) and 6.13±0.7 (D) min–1. In this and subsequent figures, error bars represent SEM of measurements, but, due to the small variability, are not always visible.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646888&req=5

pone-0063028-g001: Effects of TRAM-34 on rat CYP Activity.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 (A), 0.14±0.02 (B), 0.68±0.11 (C) and 6.13±0.7 (D) min–1. In this and subsequent figures, error bars represent SEM of measurements, but, due to the small variability, are not always visible.
Mentions: TRAM-34 was tested on CYP activities from 4 rat and 5 human isoforms. Surprisingly, concentration-dependent inhibition by TRAM-34 was seen with 3 rat CYP isoforms. TRAM-34 potently inhibited CYP2C6 and CYP2B1 (IC50 = 2.9 µM and 3.0 µM respectively, Figs. 1A and 1D). The drug showed weaker inhibition on CYP2C11 with an IC50 value of 12.6 µM (Fig. 1B). Clotrimazole, used as a positive control, was a potent inhibitor of CYP2C6, CYP2B1, and CYP2C11, as expected. TRAM-34 showed no inhibition at concentrations up to 80 µM on CYP1A2r (Fig. 1C). Clotrimazole only partly inhibited CYP1A2r activity (Fig. 1C). Other inhibitors (fluvoxamine and miconazole) did not inhibit CYP1A2r activity up to 80 µM (not shown).

Bottom Line: However, previously published work has only characterized the effects of TRAM-34 on a single CYP isoform.TRAM-34 also had both stimulatory and inhibitory effects on human CYP3A4 activity, depending on the substrate used.These results show that low micromolar concentrations of TRAM-34 can inhibit several rat and human CYP isoforms, and suggest caution in the use of high concentrations of this drug as a selective IKCa channel blocker.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York, United States of America.

ABSTRACT
TRAM-34, a clotrimazole analog characterized as a potent and selective inhibitor of intermediate-conductance, calcium-activated K(+) (IKCa) channels, has been used extensively in vitro and in vivo to study the biological roles of these channels. The major advantage of TRAM-34 over clotrimazole is the reported lack of inhibition of the former drug on cytochrome P450 (CYP) activity. CYPs, a large family of heme-containing oxidases, play essential roles in endogenous signaling and metabolic pathways, as well as in xenobiotic metabolism. However, previously published work has only characterized the effects of TRAM-34 on a single CYP isoform. To test the hypothesis that TRAM-34 may inhibit some CYP isoforms, the effects of this compound were presently studied on the activities of four rat and five human CYP isoforms. TRAM-34 inhibited recombinant rat CYP2B1, CYP2C6 and CYP2C11 and human CYP2B6, CYP2C19 and CYP3A4 with IC50 values ranging from 0.9 µM to 12.6 µM, but had no inhibitory effects (up to 80 µM) on recombinant rat CYP1A2, human CYP1A2, or human CYP19A1. TRAM-34 also had both stimulatory and inhibitory effects on human CYP3A4 activity, depending on the substrate used. These results show that low micromolar concentrations of TRAM-34 can inhibit several rat and human CYP isoforms, and suggest caution in the use of high concentrations of this drug as a selective IKCa channel blocker. In addition, in vivo use of TRAM-34 could lead to CYP-related drug-drug interactions.

Show MeSH
Related in: MedlinePlus