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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.

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Effects of TRAM-34 on human CYP Activity.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 (A–D) and fluvoxamine (C) 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 (A), 0.13±0.003 (B), 4.26±0.09 (C) and 3.81±0.32 (D) min−1.
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pone-0063028-g002: Effects of TRAM-34 on human CYP Activity.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 (A–D) and fluvoxamine (C) 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 (A), 0.13±0.003 (B), 4.26±0.09 (C) and 3.81±0.32 (D) min−1.

Mentions: TRAM-34 also showed concentration-dependent inhibition of some human CYP isoforms. The drug potently inhibited CYP2C19 and CYP2B6 (IC50 = 1.8 µM and 0.9 µM respectively, Figs. 2A and 2D). Clotrimazole, used as a positive control for CYP2C19 and CYP2B6, was a potent inhibitor of enzyme activities, as expected. TRAM-34 (up to 80 µM) showed little to no inhibition on CYP19A1h or CYP1A2h (Fig. 2B and 2C). Ketoconazole was a potent inhibitor of CYP19A1h activity, as expected. Also, unlike the results with CYP1A2r, fluvoxamine was a potent inhibitor of CYP1A2h (Fig. 2C).


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 human CYP Activity.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 (A–D) and fluvoxamine (C) 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 (A), 0.13±0.003 (B), 4.26±0.09 (C) and 3.81±0.32 (D) min−1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646888&req=5

pone-0063028-g002: Effects of TRAM-34 on human CYP Activity.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 (A–D) and fluvoxamine (C) 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 (A), 0.13±0.003 (B), 4.26±0.09 (C) and 3.81±0.32 (D) min−1.
Mentions: TRAM-34 also showed concentration-dependent inhibition of some human CYP isoforms. The drug potently inhibited CYP2C19 and CYP2B6 (IC50 = 1.8 µM and 0.9 µM respectively, Figs. 2A and 2D). Clotrimazole, used as a positive control for CYP2C19 and CYP2B6, was a potent inhibitor of enzyme activities, as expected. TRAM-34 (up to 80 µM) showed little to no inhibition on CYP19A1h or CYP1A2h (Fig. 2B and 2C). Ketoconazole was a potent inhibitor of CYP19A1h activity, as expected. Also, unlike the results with CYP1A2r, fluvoxamine was a potent inhibitor of CYP1A2h (Fig. 2C).

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